Methods and compositions for treating and preventing viral infections

ABSTRACT

A method of treating or preventing a systemic viral infection in a mammal by administering a pharmaceutically acceptable composition selected from the group consisting of squalamine, an active isomer thereof, and an active analogue thereof, via a dosing regimen that delivers effective antiviral concentrations of squalamine. Also compositions for achieving the systemic antiviral effect.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. Provisional Patent ApplicationNo. 61/255,394, filed on Oct. 27, 2009.

FIELD OF THE INVENTION

This invention relates to methods of preventing and/or treating humanand animal viral infections. The method comprises administeringsqualamine or a derivative thereof to a subject in need, which resultsin altering the lipid composition of the membranes of the tissues of thetreated subject to create a state refractory to viral infection.

BACKGROUND OF THE INVENTION

A. Background Regarding Squalamine

Chemically squalamine presented a structure never before seen in naturethat being a bile acid coupled to a polyamine (spermidine):

The discovery of squalamine, the structure of which is shown above, wasreported by Michael Zasloff in 1993 (U.S. Pat. No. 5,192,756).Squalamine was discovered in various tissues of the dogfish shark(Squalus acanthias) in a search for antibacterial agents. The mostabundant source of squalamine is in the livers of Squalus acanthias,though it is found in other sources, such as lampreys (Yun et al.,“Identification of Squalamine in the Plasma Membrane of White BloodCells in the Sea Lamprey,” Petromyzon marinus,” J. Lipid Res., 48(12):2579-2586 (2007)).

Numerous studies later demonstrated that squalamine exhibits potentantibacterial activity in vitro (Salmi, Loncle et al. 2008).Subsequently, squalamine was discovered to exhibit antiangiogenicactivity in vitro and upon administration to animals (Sills, Williams etal. 1998; Yin, Gentili et al. 2002). As a consequence, squalamine hasbeen evaluated in disease states known to be associated withpathological neovascularization, such as cancer (Sills, Williams et al.1998; Schiller and Bittner 1999; Bhargava, Marshall et al. 2001;Williams, Weitman et al. 2001; Hao, Hammond et al. 2003; Herbst, Hammondet al. 2003; Sokoloff, Rinker-Schaeffer et al. 2004), and vasculardisorders of the eye, including macular degeneration (US2007/10504A12007), retinopathy of prematurity (Higgins, Sanders et al. 2000;Higgins, Yan et al. 2004; US2007/10504A1 2007), cornealneovascularization (Genaidy, Kazi et al. 2002) and diabetic retinopathy(US2007/10504A1 2007).

The utility of squalamine as an anti-infective has been demonstrated invitro against bacteria and fungi (Moore, Wehrli et al. 1993; Rao,Shinnar et al. 2000; Salmi, Loncle et al. 2008). Squalamine is acationic amphipathic substance exhibiting an affinity for membranescomposed of anionic phospholipids (Selinsky, Zhou et al. 1998; Selinsky,Smith et al. 2000). Like other such agents, including magainin andcationic antimicrobial peptides, squalamine is believed to exertantimicrobial action by interacting electrostatically with the membranesof target microorganisms, which generally display anionic phospholipidson the membrane surface exposed to the environment, subsequentlydisturbing their functional integrity, and causing death of the targetedmicrobe (Sills, Williams et al. 1998; Zasloff 2002; Salmi, Loncle et al.2008).

To date, squalamine has not been reported to display efficacy as ananti-infective in a living animal. In no published patent application orissued patent has such evidence been reported (U.S. Pat. Nos. 5,192,756;5,637,691; 5,721,226; 5,733,899; 5,763,430; 5,792,635; 5,795,885;5,840,740; 5,840,936; 5,847,172; 5,856,535; 5,874,597; 5,994,336;6,017,906; 6,143,738; 6,147,060; 6,388,108; 6,596,712; U.S. PatentPublication No. 2005/0261508A1 2005; U.S. Pat. No. 6,962,909; U.S.Patent Publication No. 2006/0166950A1 2006; U.S. Patent Publication No.2006/0183928A1 2006; U.S. Patent Publication No. 2007/10504A1 2007).

Recent studies have revealed that squalamine is inactivated by theconcentrations of ionized calcium and magnesium present in mammalianblood, preventing squalamine from exerting its antimicrobial activity inthe setting of systemic bacterial, fungal, or protozoan infections(Salmi, Loncle et al. 2008).

Most studies of mechanism of squalamine have focused on the effects ofsqualamine on properties of the endothelial cell. The compound has beenshown to inhibit many downstream effects stimulated by diversegrowth-factors (VEGF, thrombin, FGF) including cellular proliferation,cellular migration, vascular tube formation, sodium-proton anti-porteractivation. (Sills et al., “Squalamine inhibits angiogenesis and solidtumor growth in vivo and perturbs embryonic vasculature,” Cancer Res 58,2784-92 (1998); Li et al., “Squalamine and cisplatin block angiogenesisand growth of human ovarian cancer cells with or without HER-2 geneoverexpression,” Oncogene 21, 2805-14 (2002); Akhter et al.,“Squalamine, a novel cationic steroid, specifically inhibits thebrush-border Na+/H+ exchanger isoform NHE3,” Am J Physiol 276, C136-44(1999); and Williams et al., “Squalamine treatment of human tumors innu/nu mice enhances platinum-based chemotherapies,” Clin Cancer Res 7,724-33 (2001)).

No mention of squalamine's use as a systemic antimicrobial agent, forexample, appears in a recent patent application (U.S. Patent PublicationNo. 2007/10504A1), which describes a favored salt form of squalamine fortherapeutic administration, and which addresses the utility ofsqualamine as a systemic agent in the treatment of disorders ofneovascularization and cancer.

To date, no published data describe or support the efficacy ofsqualamine in treating or preventing a systemic viral infection in ananimal. It has been reported in a patent application that squalaminecould inhibit the infectivity of HIV and HSV in tissue culture(WO96/08270). However, it was not reported at that time, nor until theinvention disclosed herein, that squalamine could exhibit antiviralactivity when administered systemically to an animal. In the experimentsdescribed in WO96/08270, squalamine was conceived as a component of atopical agent to be used as a “chemical condom”, acting as amicrobicide, and capable of rapidly inactivating HIV or HSV on contactby disrupting the outermost membranous envelopes of the viruses. Thus,the antiviral properties of squalamine observed in vitro were believedto result from direct disruption of the viral membrane, via a mechanismanalogous to that proposed for its antibacterial activity. The potentialuse of squalamine for the topical prevention of sexually transmitteddiseases such as HIV, Herpes simplex, and Neisseria gonorrhea waspresented at the 1995 ICAAC conference (MacDonald 1995). Thus,squalamine was proposed to have utility as an advanced form of“disinfectant,” to be applied to a mucosal surface in some formulationand thereby prevent viable virus from gaining access to the epithelialsurfaces of the genitourinary tract.

Squalamine has been shown to inhibit a specific isoform of thesodium-hydrogen exchanger (“NHE-3”), a protein that plays a role innumerous cellular processes that involve the control of intracellularhydrogen ions (Akhter, Nath et al. 1999). As a consequence of thisactivity, it was proposed that squalamine might find utility in treatingdiseases, including viral infections, where NHE3 played a critical role,and where its inhibition (by squalamine) could be effected (see e.g.,U.S. Pat. No. 6,962,909). It has been proposed that squalamine could beused to treat viral infections should it be known that a specific virusinfected a target cell expressing an NHE sensitive to inhibition (NHE-3in the case of squalamine), and that the specific NHE played a criticalrole in the cellular homeostasis of that cell type, and that the virusin question naturally infected that cell type in the course of a diseaseprocess (U.S. Pat. No. 6,962,909). To date, however, no example of anNHE-3 dependent viral infection has been reported in the literature, norhas any known NHE-3 inhibitor been shown to exhibit antiviral activityin an animal, including squalamine. Furthermore the viruses demonstratedto be inactivated in vitro by squalamine, namely HIV and HSV(WO96/08270) are now known to infect cells via a pathway that is “pHindependent”, in the sense that inhibitors of pH homeostasis do notinfluence infectivity (Pelkmans and Helenius 2003).

1436 is an aminosterol, isolated from the dogfish shark, structurallyrelated to squalamine (U.S. Pat. No. 5,840,936; Rao, Shinnar et al.2000). Aminosterol 1436 exhibits antiviral activity against HIV intissue culture (U.S. Pat. No. 5,763,430) via a mechanism proposed toinvolve inhibition of a lymphocyte-specific NHE by 1436, resulting insuppression of cytokine responsiveness, and subsequent depression of thecapacity of the lymphocyte to support HIV replication (U.S. Pat. No.5,763,430). Aminosterol 1436, however, has an additional pharmacologicalproperty, not shared with squalamine, namely potent appetite suppressionand promotion of dose-dependent weigh loss (U.S. Pat. No. 6,143,738;Zasloff, Williams et al. 2001; Ahima, Patel et al. 2002). Administrationof Aminosterol 1436 to animals at doses that would achieve tissueconcentrations of Aminosterol 1436 speculated to exert an antiviralbenefit cause profound weight loss and suppression of food intake anddeath due to starvation (Zasloff, Williams et al. 2001; Ahima, Patel etal. 2002).

Recent patents have been issued describing squalamine like compoundswith potent antibacterial activity, but no mention is made of theirutility as antiviral agents (U.S. Pat. Nos. 5,834,453; 6,017,906).Indeed, the potential value of squalamine and its analogs as systemicagents has been questioned due to the extensive binding to albuminexhibited by these compounds (U.S. Pat. No. 5,834,453).

Squalamine in its intravenous form, squalamine lactate, is in theprocess of being tested as a treatment of fibrodysplasia ossificansprogressiva, a rare disease where connective tissue will ossify whendamaged. (Genesis, A., “Squalamine trial for the treatment offibrodysplasia ossificans progressiva initiated”, Angiogenesis Weekly,8:45 (2002).) Squalamine is also undergoing trials for treatment ofnon-small cell lung cancer (stage I/IIA) as well as general phase Ipharmacokinetic studies. (Herbst et al., “A Phase I/IIA Trial ofContinuous Five-Day Infusion of Squalamine Lactate (MSI-1256F) PlusCarboplatin and Paclitaxel in Patients with Advanced Non-Small Cell LungCancer 1”, Clinical Cancer Research, 9:4108-4115 (2003); Hao et al., “APhase I and Pharmacokinetic Study of Squalamine, an AminosterolAngiogenesis Inhibitor”, Clin Cancer Res., 9(7): 2465-2471 (2003).) In2005, the Food and Drug Administration granted squalamine Fast Trackstatus for approval for treatment of age-related macular degeneration.(CATE: California Assistive Technology Exchange”, California AssistiveTechnology Exchange,http://cate.ca.gov/index.cfm?a=Resources&p=News&article=176, Retrieved2009-03-31.) However, Genaera Corporation discontinued trials for theuse of squalamine in treating prostate cancer and wet age-relatedmacular degeneration in 2007. (“PROSTATE CANCER; Genaera DiscontinuesLOMUCIN in Cystic Fibrosis and Squalamine in Prostate Cancer Studies”,Drug Week, pp. 251. 2007-07-20; “Reports describe the most recent newsfrom Genaera Corporation”. Biotech Business Week, pp. 1540(2007-09-17).) Squalamine is also marketed under the brand nameSqualamax™ as a dietary supplement, though it has not been approved as adrug in this form and thus cannot make therapeutic claims. Squalamax™ isan unfractionated extract of shark liver, containing innumerableuncharacterized substances in addition to squalamine, itself presentbelow 0.01% of the total weight of the extract. (“Cyber Warning Letter”,Center for Drug Evaluation and Research (2002-05-06),http://www.fda.gov/CDER/warn/cyber/2002/CFSANnuGen.htm; Retrieved2009-03-31.) Moreover, the dietary supplement form of squalamine is notpharmaceutical grade squalamine, which requires significantly greatermanufacturing efforts.

By 2006, over 300 patients had received squalamine in doses ranging from6-700 mg/m2/day by iv administration, in three Phase I and nine Phase IIstudies (Hao et al., “A Phase I and pharmacokinetic study of squalamine,an aminosterol angiogenesis inhibitor,” Clin Cancer Res 9, 2465-71(2003); Herbst et al., “A phase I/IIA trial of continuous five-dayinfusion of squalamine lactate (MSI-1256F) plus carboplatin andpaclitaxel in patients with advanced non-small cell lung cancer,” ClinCancer Res 9, 4108-15 (2003); Bhargava et al., “A phase I andpharmacokinetic study of squalamine, a novel antiangiogenic agent, inpatients with advanced cancers,” Clin Cancer Res 7, 3912-9 (2001); andConnolly et al., “Squalamine lactate for exudative age-related maculardegeneration,” Ophthalmol Clin North Am 19, 381-91, vi (2006). Thestudies showed that the compound exhibited an acceptable safety profileand evidence of efficacy in these early trials. In 2006 development ofsqualamine was halted for economic/strategic reasons by Genaera, and hasremained in a dormant stage since.

There is a need in the art for new treatments for viral infections.There are a wide variety of viral diseases having limited or ineffectivetreatments. The present invention addresses the problem by providing anew method of treating and/or preventing viral infections.

SUMMARY OF THE INVENTION

The present invention is directed to methods of treating and/orpreventing viral infections comprising administering a therapeuticallyeffective amount of squalamine or a derivative thereof, an isomer orprodrug of squalamine, or a pharmaceutically equivalent salt thereof toa subject, such as a mammal, in need. A “subject in need” is a human oranimal at risk of a viral infection, or which has contracted a viralinfection. Preferably, the squalamine is a pharmaceutical gradesqualamine. Preferably, the squalamine or derivative thereof is apharmaceutical grade of squalamine, and the composition can furthercomprise one or more pharmaceutically acceptable excipients. Thesqualamine or derivative thereof is present in an amount sufficient toproduce an antiviral effect.

In another embodiment, the invention encompasses methods of treatingand/or preventing viral infections comprising administering atherapeutically effective amount of an aminosterol that can inhibit theformation of actin stress fibers in endothelial cells stimulated by aligand known to induce stress fiber formation, having the chemicalstructure of Formula I:

wherein,

-   -   W is 24S—OSO₃ or 24R—OSO₃;    -   X is 3β-H₂N—(CH₂)₄—NH—(CH₂)₃—NH— or 3α-H₂N—(CH₂)₄—NH—(CH₂)₃—NH—;    -   Y is 20R—CH₃; and    -   Z is 7α or 7β-OH

In yet another embodiment of the invention, the aminosterol is aderivative of squalamine modified through medical chemistry to improvebiodistribution, ease of administration, metabolic stability, or anycombination thereof. In another embodiment, the squalamine oraminosterol is modified to include one or more of the following: (1)substitutions of the sulfate by a sulfonate, phosphate, carboxylate, orother anionic moiety chosen to circumvent metabolic removal of thesulfate moiety and oxidation of the cholesterol side chain; (2)replacement of a hydroxyl group by a non-metabolizable polarsubstituent, such as a fluorine atom, to prevent its metabolic oxidationor conjugation; and (3) substitution of various ring hydrogen atoms toprevent oxidative or reductive metabolism of the steroid ring system.

In certain embodiments of the invention, the methods compriseadministering squalamine or a derivative thereof at an effective dailydosing amount of about 0.1 to 20 mg/kg body weight. In otherembodiments, the effective amount is administered in a regimen thatachieves and maintains a tissue concentration of squalamine in bodyorgans and tissues of between about 0.1-200 μg/gram (tissue wet weight).

The composition can be administered via any pharmaceutically acceptablemethod, including but not limited to intravenously, subcutaneously,intramuscularly, topically, orally, or by inhalation.

In one embodiment of the invention (a) the composition does notdemonstrate an altered IC₅₀ or IC₉₀ (drug concentration required toinhibit viral growth by 50% or 90% respectively) over time; (b) thecomposition demonstrates an IC₅₀ or IC₉₀ which does not increase by morethan 0%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, or 30% over time; (c) thecomposition demonstrates an IC₅₀ or IC₉₀ which does not increase by anamount described in (b) over a time period selected from the groupconsisting of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 1.5 months, 2months, 2.5 months, 3 months, 3.5 months, 4 months, 4.5 months, 5months, 5.5 months, 6 months, 6.5 months, 7 months, 7.5 months, 8months, 8.5 months, 9 months, 9.5 months, 10 months, 10.5 months, 11months, 11.5 months, 12 months, 1 year, 1.5 years, 2 years, 2.5 years, 3years, 3.5 years, 4 years, 4.5 years, and 5 years; or (d) anycombination thereof.

The viral infection to be treated or prevented can be caused by anyvirus, including but not limited to, “African Swine Fever Viruses,”Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Astroviridae,Baculoviridae, Birnaviridae, Birnaviridae, Bunyaviridae, Caliciviridae,Caulimoviridae, Circoviridae, Coronaviridae, Cystoviridae, Dengue, EBV,HIV, Deltaviridae, Filviridae, Filoviridae, Flaviviridae, Hepadnaviridae(Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex,Herpes Zoster), Iridoviridae, Mononegavirus (e.g., Paramyxoviridae,Morbillivirus, Rhabdoviridae), Myoviridae, Orthomyxoviridae (e.g.,Influenza A, Influenza B, and parainfluenza), Papiloma virus,Papovaviridae, Paramyxoviridae, Prions, Parvoviridae, Phycodnaviridae,Picornaviridae (e.g. Rhinovirus, Poliovirus), Poxviridae (such asSmallpox or Vaccinia), Potyviridae, Reoviridae (e.g., Rotavirus),Retroviridae (HTLV-I, HTLV-II, Lentivirus), Rhabdoviridae, Tectiviridae,Togaviridae (e.g., Rubivirus), or any combination thereof. In anotherembodiment of the invention, the viral infection is caused by a virusselected from the group consisting of herpes, pox, papilloma, corona,influenza, hepatitis, sendai, sindbis, vaccinia viruses, west nile,hanta, or viruses which cause the common cold. In another embodiment ofthe invention, the condition to be treated is selected from the groupconsisting of AIDS, viral meningitis, Dengue, EBV, hepatitis, and anycombination thereof.

In another embodiment of the invention, the condition to be treated is achronic disease suspected to be of viral origin. For example, thecondition to be treated can be multiple sclerosis, Type I diabetes, TypeII diabetes, atherosclerosis, cardiomyopathies, Kawaski disease,aplastic anemia, etc.

The methods of the invention can further comprise administering thesqualamine or derivative thereof in combination with at least oneadditional active agent to achieve either an additive or synergisticantiviral effect. The additional active agent can be administeredconcomitantly, as an admixture, separately and simultaneously orconcurrently, or separately and sequentially. For example, theadditional active agent can be: (a) an antiretroviral agent; (b)nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs); (c)non-nucleoside reverse transcriptase inhibitors (NNRTIs); (d) nucleotideor nucleoside analogues; (e) protease inhibitors (PIs); (f) drugs basedon “antisense” molecules; (g) ribozyme antivirals; (h) assemblyinhibitors; (i) release phase inhibitors; (j) drugs which stimulate theimmune system, such as interferons and synthetic antibodies; (k) fusioninhibitors/gp41 binders; (l) fusion inhibitors/chemokine receptorantagonists; (m) integrase inhibitors; (n) hydroxyurea-like compounds;(o) inhibitors of viral integrase; (p) inhibitors of viral genomenuclear translocation; (q) inhibitors of HIV entry; (r) nucleocapsidzinc finger inhibitors; (s) targets of HIV Tat and Rev; (t)pharmacoenhancers; (u) cytokines; (v) lymphokines; (w) ananti-inflammatory agent; or (x) any combination thereof.

In one embodiment of the invention, described are antiviral compositionscomprising at least one squalamine, a squalamine derivative, asqualamine isomer or prodrug, or a pharmaceutically equivalent saltthereof. The compositions can further comprise at least one antiviralimmunological adjuvant. Examples of antiviral immunological adjuvantsinclude, but are not limited to corticosteroids, alpha-interferon, etc.

In yet another embodiment of the invention, the composition can furthercomprise at least one antigen capable of eliciting an immune response.For example, the antigen can be a viral or prion antigen.

In another embodiment of the invention, combination methods of treatingor preventing a viral infection are described. The combination methodscomprise:

(1) administering a therapeutically effective amount of squalamine, aderivative, a squalamine isomer or prodrug, or a pharmaceuticallyequivalent salt thereof to a subject in need; and (2) administering aconventional antiviral drug. The squalamine composition and conventionalantiviral drug can be administered sequentially or simultaneously. Ifsqualamine or a conventional antiviral drug are administeredsequentially, either squalamine or the conventional antiviral drug canbe administered first.

Also described are compositions comprising (1) at least one squalaminecompound, a squalamine isomer or prodrug, or a pharmaceuticallyequivalent salt thereof to a subject in need; and (2) at least oneconventional antiviral drug. The compositions can additionally compriseat least one pharmaceutically acceptable excipient or carrier.

Both the foregoing summary of the invention and the following briefdescription of the drawings and the detailed description of theinvention are exemplary and explanatory and are intended to providefurther details of the invention as claimed. Other objects, advantages,and novel features will be readily apparent to those skilled in the artfrom the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows a picture of a cell before (FIG. 1A) and after (FIG. 1B)exposure to squalamine. FIG. 1A shows the net negative charge at thecell surface (i.e., green circle) and FIG. 1B shows the change in cellstructure following exposure to squalamine. Specifically, squalamineintegrates into the cellular membrane, profoundly altering the overallcharge of that membrane, and causing displacement of key proteins boundto the membrane through electrostatic interactions and required foractin remodeling to occur.

FIG. 2: Shows microscopic visualization following transfection of a RAW264.7 macrophage line with engineered recombinant vectors to generatecells that expressed two peptide probes, each linked to a red (FIGS. 2Aand 2D) or green (FIGS. 2B and 2E) fluorescent protein. “Trunc Cat Tail”(GFP-ARDGRRRRRRARARCVIM) is a highly cationic probe, that associateswith the plasma membrane through electrostatic forces. “H-Ras” (RFP—fulllength H-Ras) is a member of the Ras family of proteins that associateswith the plasma membrane predominantly through hydrophic forces. Priorto exposure of these cells to squalamine, both H-Ras and Trunc Cat Tailcan be seen associated with the plasma membrane (FIGS. 2A, 2B, and 2C).Following the addition of squalamine (10 micromolar) to the culturemedium in which the cells are bathed, the True Cat Tail probe isdisplaced into the cytoplasm, while the H-Ras probe remains associatedwith the membrane (FIGS. 2D, 2E, and 2F).

FIG. 3: Shows the results of a study exploring squalamine's mechanism ofaction using a cell which expresses two probes, with the effects oftreatment noted by comparing the cell before and after exposure tosqualamine. The RAW264.7 cell line has been engineered to express a redtagged fluorescent protein (“Lact-C2”) that binds avidly tophosphatidylserine. Lact-C2 binds to phosphatidylserine (a negativelycharged phospholipid) through highly specific interactions that dependon the “shape” of the phospholipid, rather than its electric charge. Inaddition the cell line also expresses a green tagged fluorescentcationic fragment (“R-pre”). R-pre binds to phosphatidylserine as aconsequence of electrostic interactions, the strongly positive peptideattracted to the strong negative charges present on the head ofphosphatidyl serine. FIG. 3A shows the cells before exposure tosqualamine and FIG. 3B shows the cells following exposure to squalamine.As seen in FIG. 3A, before addition of squalamine, both probes are seento be associated with the plasma membrane of the cells, as expected.Following exposure of these cells to squalamine (80 μM, 30 minutes),R-pre was displaced from its residence on the plasma membrane to otherareas within the cell's interior. In contrast, exposure of these cellsto squalamine did not alter the localization of Lact-C2. This experimentsupports the hypothesis that squalamine, a positively charged molecule,neutralizes the negatively charged phosphplipids upon entry into themembrane, and can, as a consequence, cause the displacement of membraneproteins bound by virtue of electrostatic interactions.

FIG. 4: Shows the results of an in vivo test to determine theeffectiveness of squalamine against Yellow Fever in Syrian hamsters.Squalamine was administered to infected Syrian hamsters at 0.7, 2.5, and7.7 mg/kg once daily achieving an antiviral effect. At 7.7 mg/kg/day,60% survival was observed, similar to the survival achieved withadministration of ribivarin, 50 mg/kg/day, and compared to the 20%survival seen in animals receiving a placebo (saline).

FIG. 5: Shows the results of an in vivo test to determine theeffectiveness of squalamine against Yellow Fever in Syrian hamsters in ahead-to-head comparison with the antiviral drug ribavirin. Squalamine at15 mg/kg was administered subcutaneously daily, while ribavirin wasadministered once daily i.p. at either 3.2, 10, and 32 mg/kg. Squalaminewas the most effective treatment, with 70% of the animals surviving,compared with about 10% of those receiving vehicle. Ribavirin was lesseffective, the maximal dose achieving a survival of 40%.

FIG. 6: Shows the results of an in vivo test to determine theeffectiveness of squalamine against an established Yellow Fever inSyrian hamsters. Squalamine treatment is shown to cure a lethalinfection when administered 1 or 2 days after viral administration.

FIG. 7: Shows the results of an in vivo test to determine theeffectiveness of squalamine against Cytomegalovirus infection in themouse. Squalamine, administered at 10 mg/kg/day i.p., is shown toachieve a reduction of viral titers in spleen and liver to undetectablelevels in infected animals.

FIG. 8: Shows the results of an in vivo test to determine theeffectiveness of squalamine against Eastern Equine Encephalitis virus inSyrian hamsters. Squalamine administered at 10 mg/kg/day, s.c., is shownto increase survival, compared with a vehicle control.

FIG. 9: Shows the results of an in vivo test to determine theeffectiveness of squalamine against Eastern Equine Encephalitis virus inSyrian hamsters. Squalamine administered at 10 mg/kg/day s.c. is shownto significantly reduce viremia compared with a vehicle control, in theexperiment described in FIG. 8.

FIG. 10: Shows the results of an in vitro study to assay the antiviralactivity of squalamine against Dengue virus. Human microvascularendothelial cells were exposed to Dengue virus in the presence ofincreasing concentrations of squalamine. Viral infection was monitoredby immunofluorescent analysis of the Dengue E protein. At 46 ug/ml,squalamine achieved an inhibition of viral infection of about 80%, with100% inhibition observed at 62 ug/ml.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods of treating and/orpreventing viral infections comprising administering a therapeuticallyeffective amount of squalamine, an isomer or prodrug of squalamine, asqualamine derivative, or a pharmaceutically equivalent salt thereof toa subject in need. A “subject in need” is a human or animal at risk of aviral infection, or which has contracted a viral infection.

A variant or derivative of squalamine may have one or more chemicalmodification which do not modify the antiviral activity of squalamine. A“variant” or “derivative” of squalamine is a molecule in whichmodifications well known in the art of medicinal chemistry to “mimic”the original spatial and charge characteristics of a portion of theoriginal structure have been introduced to improve the therapeuticcharacteristics of squalamine. In general, such modifications areintroduced to influence metabolism and biodistribution. Examples of suchvariants or derivatives include, but are not limited to, (1)substitutions of the sulfate by a sulfonate, phosphate, carboxylate, orother anionic moiety chosen to circumvent metabolic removal of thesulfate moiety and oxidation of the cholesterol side chain; (2)replacement of an hydroxyl group by a non-metabolizable polarsubstituent, such as a fluorine atom, to prevent its metabolic oxidationor conjugation; and (3) substitution of various ring hydrogen atoms toprevent oxidative or reductive metabolism of the steroid ring system. Asused herein, the term “squalamine” is intended to encompass squalamineand variants or derivatives thereof.

In another embodiment, the invention encompasses methods of treatingand/or preventing viral infections comprising administering atherapeutically effective amount of an aminosterol that can inhibit theformation of actin stress fibers in endothelial cells stimulated by aligand known to induce stress fiber formation, having the chemicalstructure of Formula I:

wherein,

-   -   W is 24S—OSO₃ or 24R—OSO₃;    -   X is 3β-H₂N—(CH₂)₄—NH—(CH₂)₃—NH— or 3α-H₂N—(CH₂)₄—NH—(CH₂)₃—NH—;    -   Y is 20R—CH₃; and    -   Z is 7α or 7β-OH

To date, a hypothesis that explains the diversity of squalamine'seffects has not been reported. While not wishing to be bound by anyparticular theory, the inventor believes that squalamine exerts itseffects by interrupting a key step in the pathways involved in actindynamics, which it achieves by an unprecedented mechanism. Squalaminedoes so by integrating in the cellular membrane, profoundly altering theoverall charge of that membrane, and causing displacement of keyproteins bound to the membrane through electrostatic interactions andrequired for actin remodeling to occur. Thus, upon entry into a cell,squalamine profoundly alters the behavior of the circuitry involved incontrol of the actin cytoskeleton. Most viruses must exert control overthe actin cytoskeleton to gain entry into the cell they target. Thisalteration by squalamine effectively “closes the door” to viral entryinto the cell. This is because a substance that interrupts the actinremodeling circuitry of a target cell utilized by a virus for infectionmakes the cell “resistant” so long as the disruptive effects persist.

The basic mechanism of action of squalamine should be operative in anycell into which squalamine can gain entry. Thus, squalamine can preventviral infection of any cell into which squalamine can gain entry.Moreover, because of the broad tissue distribution of squalamine, thecompound can alter the virulence of a virus by interfering with itsinfectivity of any number of tissues in the animal, a “whole animal”effect that might be missed in a simple cellular screen. Indeed,squalamine represents a class of antiviral that achieves its therapeuticeffect by creating a state of viral resistance within the treatedanimal, rather than by directly targeting a viral enzyme or protein.During this period of squalamine resistance, viral particles, unable toinfect tissues, would be cleared and destroyed by the cellularmechanisms that are normally engaged to dispose of particles of theirsize and composition (i.e., phagocytic destruction by neutrophils,macrophages, and the reticuloendothelial system). Furthermore, as aconsequence of the mechanism proposed for the antiviral activity ofsqualamine, which involves inhibition of cellular circuitry used byviruses to remodel the actin cytoskeleton to permit invasion, squalaminewould be expected to exhibit a very broad spectrum of activity, coveringviruses of all classes, regardless of their genome composition (RNA vsDNA viruses).

In the case of squalamine, the “resistance” state should last as long asthe compound persists in circulation, that being several hours. Based onthe known pharmacokinetics of squalamine in rodents, dogs and humans,following administration the compound should rapidly gain entry to awide range of cells, remain in intracellular sites for between minutesto hours, and eventually traffic out of the cell, unmetabolized,re-entering the circulation, to then be transported into the hepatocytevia its basolateral surface, passage through the cell and subsequentlytransported from the apical surface of the hepatocyte into the biliarytract.

FIGS. 1A and 1B show the physical changes in cell structure uponexposure to squalamine. More particularly, FIG. 1A shows a picture of acell before exposure to squalamine. The net negative charge of thecytoplasmic face of the plasma membrane at the cell surface is clearlydepicted by virtue of the adherence of the green fluorescent positivelycharged probe, which creates a green outline at the cell's periphery.After exposure to squalamine, as shown in FIG. 1B, squalamine integratesinto the cellular membrane, profoundly altering the overall charge ofthat membrane, and causing displacement of key proteins bound to themembrane through electrostatic interactions and required for actinremodeling to occur. The green cationic probe, displaced from themembrane surface as a consequence of the neutralization of the negativecharge, diffuses into the cytoplasm, filling the cell with a greencolor. It is this change in the cell structure which inhibits viralinfection of the cell. Specifically, viruses seek the negatively chargedcell surface as a “gateway” to the cell for infection. Squalamineeffectively closes the gateway by changing the charge and structure ofthe cell membrane.

Lack of Resistance: Antiviral drug resistance is a significant problemencountered with treating and preventing viral infections. Antiviralresistance means that a virus has changed in such a way that theantiviral drug is less effective in treating or preventing illnessescaused by the virus. Virally encoded drug resistance has been documentedagainst nearly all compounds with antiviral activity. Drug resistance isdefined as a reduced susceptibility to a drug in a laboratory culturesystem and is expressed as an altered IC₅₀ or IC₉₀ (drug concentrationrequired to inhibit viral growth by 50% or 90% respectively). This istermed the phenotype. This phenotype is determined by specific mutationsin the viral genome (the genotype), which leads to alterations in theviral target protein (for example, HIV reverse transcriptase) or theviral drug activator (for example, herpes simplex thymidine kinase). Thehigh rate of replication of some viruses determines that many of thesegenetic variants will already exist in untreated infected people. Thisis consequent on an inherent error rate of viral polymerases, especiallyfor RNA viruses such as HIV and influenza, which replicate the viralgenome. A wide range of viral variants, including those with mutationsassociated with drug resistance, will therefore be present. Thiscollection of variants in one person is termed the viral quasispecies,with the “fittest” virus representing the majority population. The useof an antiviral drug will provide a selective pressure for thepreferential growth of variants with a reduced susceptibility to drugsin accordance with Darwinian evolutionary principles. The emergent drugresistant virus will be the fittest in the presence of drug. Some drugresistant viruses, however, seem not to replicate as well as wild typevirus (in the absence of drug). In some cases, multiple mutations arerequired for the development of high level resistance, and insufficientsuppression of viral replication by antiviral drugs will predispose totheir sequential acquisition. Pillay et al., “Antiviral drugresistance,” Public Health Laboratory Service Antiviral SusceptibilityReference Unit, Division of Immunity and Infection, University ofBirmingham Medical School, Birmingham B15 2TT,http://www.bmj.com/content/vol317/issue7159/fulltext/supplemental/660/index.shtml,accessed on Oct. 21, 2009.

In contrast to traditional antiviral therapies, viruses are not expectedto develop resistance to squalamine. This is because unlike conventionalantiviral therapies, squalamine does not act upon a single mechanism bywhich a virus infects a cell. Rather, squalamine changes the cellstructure for a period of time during which the virus cannot infect thecell. In contrast, certain anti-HIV drugs target the CD4 receptor andother antiviral drugs target inhibition of replication. Viral variantscan circumvent each of these targeted antiviral therapies. In oneembodiment of the invention, squalamine does not demonstrate an alteredIC₅₀ or IC₉₀ (drug concentration required to inhibit viral growth by 50%or 90% respectively) over time. In other embodiments of the invention,squalamine demonstrates an IC₅₀ or IC₉₀ which does not increase by morethan 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 25%, or 30% over time. In otherembodiments of the invention, the time period over which the change inIC₅₀ or IC₉₀ (or lack thereof) is measured is 1 week, 2 weeks, 3 weeks,4 weeks, 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 3.5months, 4 months, 4.5 months, 5 months, 5.5 months, 6 months, 6.5months, 7 months, 7.5 months, 8 months, 8.5 months, 9 months, 9.5months, 10 months, 10.5 months, 11 months, 11.5 months, 12 months, 1year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5years, or 5 years.

Toxicity: Conventional antiviral agents are generally designed to targetviral specific enzymes, such as RNA and DNA polymerases, proteases, orglycosidases; as a consequence the drug inhibits the activity of theviral enzyme to a far greater extent than it does to analogous humanenzymes, required for normal cellular functioning. In many instancestoxicity develops as a consequence of the residual activity of the agenttowards the analogous enzymes of the host. The experience collected todate involving the administration of squalamine to humans suggests thatthe compound has an acceptable therapeutic index, a property thatfurther enhances the utility of the invention disclosed herein.

I. Definitions

The following definitions are provided to facilitate understanding ofcertain terms used throughout this specification.

As used herein, “therapeutic activity” or “activity” may refer to anactivity whose effect is consistent with a desirable therapeutic outcomein humans, or to desired effects in non-human mammals or in otherspecies or organisms. Therapeutic activity may be measured in vivo or invitro. For example, a desirable effect may be assayed in cell culture.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill in the art given the context in which it is used, “about”will mean up to plus or minus 10% of the particular term.

As used herein, the phrase “therapeutically effective amount” shall meanthe drug dosage that provides the specific pharmacological response forwhich the drug is administered in a significant number of subjects inneed of such treatment. It is emphasized that a therapeuticallyeffective amount of a drug that is administered to a particular subjectin a particular instance will not always be effective in treating theconditions/diseases described herein, even though such dosage is deemedto be a therapeutically effective amount by those of skill in the art.

II. Mechanism Of Squalamine'S Antiviral Activity

A. The Relationship Between Squalamine And Membrane ElectrostaticPotential

Squalamine is a cationic (net positively charged) aminosterol. Itpossesses a negatively charged sulfate group on its cholesterol sidechain that contributes a single negative charge; however, it alsopossesses the polyamine spermidine attached to the opposite side of themolecule. This moiety has three positively charged amino groups (atphysiological pH). Overall, therefore, squalamine exhibits a netpositive charge of 2, and these charges are localized to a specificregion of the molecule, that being the polyamine. Because of its netpositive charge and amphilicity, squalamine partitions into membranes ofappropriate composition and interacts electrostatically with negativelycharged phospholipids within the membrane. Thus, upon partitioning intoa membrane, squalamine is expected to reduce the net negative charge ofthe membrane. Thus, the present invention describes the properties of acationic lipid (squalamine) which permits safe and effectivemodification of the cellular membranes of a tissue or organ in such afashion as to reduce the capacity of the tissues or organs to beinfected by a virus. Additionally, the invention describes the discoveryof specific cellular transporters that either restrict or facilitate thepassage of an aminosterol into a tissue or organ, knowledge which candirect the application of the invention to specific viral infections.

It has been only recently appreciated that membrane surfaces presentwithin the interior of animal cells exhibit a net negative surfacecharge, also referred to as “a net negative electrostatic potential”.This net negative charge results from the presence of specific anionicphospholipids that comprise these membrane surfaces. For example, theanionic phospholipid, phosphatidylserine, is the most abundant anionicphospholipid in animal cells, is present on the inner surface of theplasma membrane of animal cells, and is the principal lipid responsiblefor the negative electrostatic charge of the inner layer of the plasmamembrane (McLaughlin and Murray 2005; Yeung, Terebiznik et al. 2006;Steinberg and Grinstein 2008; Yeung, Gilbert et al. 2008). Similarly,phosphatidylserine is present in other intracellular membranouslocations, such as the endosomes and the Golgi apparatus, but in lesserproportions than observed for the plasma membrane, and thus, conferringa weaker overall negative charge on these internal membranes. Anexcellent review on the role of anionic phospholipids in establishingthe negative electrostatic potential of cellular membranes has beenrecently published (Steinberg and Grinstein 2008).

The negative electrostatic potential of intracellular membranes is nowbelieved to play a major role in the physical positioning of manyintracellular proteins that physically associate with intracellularmembranes (McLaughlin and Murray 2005; Yeung, Terebiznik et al. 2006;Steinberg and Grinstein 2008; Yeung, Gilbert et al. 2008). It has beendiscovered that many proteins involved in important cellular functionsare themselves positively charged, and as a consequence of electrostaticinteractions, are directed to negatively charged membranes where theyare positioned appropriately to execute their functions. Of particularnote are the many small GTPases involved in intracellular signaling. Inparticular, the RhoGTPases, including Rac and Cdc 42, which play acentral role in the dynamics of the actin cytoskeleton of all eukaryoticcells, are tethered to the inner surface of the plasma membrane viaelectrostatic interactions (Kelly 2005; Yeung, Terebiznik et al. 2006;Yeung, Gilbert et al. 2008).

Squalamine can reduce the negative electrostatic potential of thecytoplasmic surface of a cell and displace proteins associated viaelectrostatic forces. Specifically, based on the cationic character ofsqualamine and the importance of electrostatics in the association ofkey proteins with the inner surface of the plasma membrane, it isexpected that upon exposure of a cell to squalamine, and its subsequententry into the plasma membrane, proteins associated with the cytoplasmicsurface via electrostatic interactions will be displaced and releasedinto the cellular cytoplasm.

As demonstrated in the Examples below, squalamine can reduce the netelectrostatic potential of cellular membranes into which it enters.Remarkably, it does so without disrupting the physical integrity of themembranes into which it integrates. This striking property of squalamineis likely a result of the manner in which squalamine positions itself onthe membrane. Analysis of its structure would suggest that squalaminelies superficially on the surface of the membrane, interactingelectrostatically with the lipid headgroups at the aqueous interface,rather than by burying itself within the lipid phase. As a consequence,squalamine can displace proteins normally positioned via electrostaticinteractions on the plasma membrane and other membranes to whichsqualamine is known to traffic without directly disrupting the physicalintegrity of the membrane.

As demonstrated in the literature, reduction in the electrostaticpotential of the plasma membrane of the magnitude required to displaceTrunc Cat Tail and R-pre (see e.g., Example 1) should be sufficient tocause displacement of many of the GTPases known to be associated withthe plasma membrane. In particular, it is reasonable to highlight Rac1and Cdc 42, two GTPases known to be anchored through phosphatidylserinebased interactions (Kelly 2005; Finkielstein, Overduin et al. 2006;Yeung, Terebiznik et al. 2006; Yeung, Gilbert et al. 2008). These RhoGTPases play a central role in the dynamics of the actin cytoskeleton ofall eukaryotic cells. These dynamics come into play when cells migrate,during endocytosis, in the processes of membrane ruffling, filopodiaformation, and so on.

Indeed, the results of Example 1 suggest a mechanism to explain certaininhibitory effects of squalamine on the endothelial cell, such asmigration, growth factor dependent stress fiber formation, and theformation of focal adhesion contacts, each an actin based process(Sills, Williams et al. 1998; Williams, Weitman et al. 2001).

Relevant to the present invention, the Rho GTPases, as key components inthe dynamic remodeling of the actin cytoskeleton, are known to becritically involved in numerous events in the life cycle of most, if notall, known animal viruses (Pelkmans, Fava et al. 2005; Mercer andHelenius 2008), including Vaccinia and small pox, West Nile virus,influenza, yellow fever, dengue, Adenoviruses, Rubella, and HIV.Displacement of these RhoGTPases from the plasma membrane, resulting intheir functional inactivation, would necessarily result in perturbationof the life cycle of a virus, and thereby reduce its infectivity. Inthis state, the squalamine treated cell would appear resistant towardsall viruses that required these Rho GTPases for infection.

B. Viral Infection, Anionic Phospholipids, and Electrostatic Potential

Viruses must deliver their genomes into a target cell. They accomplishthis ultimately by fusing with the plasma membrane of the cell. Virusessurrounded by membranes can fuse directly with the membrane of a cell.Alternatively, both enveloped and non-membrane enclosed viruses can beengulfed by an endocytic process. Escape from the endosomal compartmentrequires a fusion event between the viral envelope or its membrane topermit the viral genome to gain entry into the cell's interior. Theseevents in the life cycle of all viruses are known to engage cellularmachinery involved in actin cytoskeletal remodeling. Indeed, viralinfectivity is now known to be sensitive to inhibition by agents thatdisrupt the normal functioning of actin remodeling machinery. Byaltering the electrostatic potential of the inner surface of a cell, andconsequently disturbing the association of proteins required for thenormal dynamics of actin cytoskeletal remodeling, squalamine shouldinfluence actin dynamics involved in efficient viral infection.

Viral entry and infection, involving membrane fusion, viral uptake andinternalization, appear to be exceedingly sensitive to the presence ofadequate amounts of cellular anionic phospholipid, especiallyphosphatidylserine (Coil and Miller 2005; Coil and Miller 2005; Mercerand Helenius 2008). Indeed, most, if not all cells, appear to haveinsufficient phosphatidylserine to support maximal viral infectivity(Coil and Miller 2004; Coil and Miller 2005; Coil and Miller 2005).Hence, adding exogenous phosphatidylserine to a wide range of cell typesincreases the viral infectivity of a wide range of virus. Conversely,cells that are genetically deficient in phosphatidylserine (and requireexogenous phosphatidylserine for survival) appear to become less capableof supporting viral infection as total cellular stores ofphosphatidylserine decrease (Kuge, Akamatsu et al. 1989). Precisely whythe cellular content of phosphatidylserine should be rate limiting formany (if not all) viruses is not well understood (Coil and Miller 2004;Mercer and Helenius 2008). However, since squalamine is known to complexwith phosphatidylserine in membranes, the presence of this aminosterolwould effectively reduce the free concentration of phosphatidylserinewithin the cell, effectively depleting a cellular lipid critical forviral infectivity.

C. Squalamine is a Substrate for the Organic Cation Transporters Oct 1-3

Squalamine, an ionic compound with a strong negative charge (provided bythe sulfate moiety) and three strong positive charges (provided by theprotonated spermidine), would not be expected to enter cells freely bypermeating or diffusing through the plasma membrane. Molecules such asthese generally enter cells via interactions with transporting proteinssituated on the plasma membrane. Furthermore, although squalamine couldinteract directly with anionic phospholipids and subsequently integrateinto the cellular membrane, cells do not normally expose anionicphospholipids on the outer leaflet of the plasma membrane, the surfaceexposed to the “outside world”. As pointed out above, it is the innersurface of the plasma membrane that normally bears a negativeelectrostatic potential.

To date, no published data exist that describes the transporters thatthe aminosterols must utilize to enter cells. Knowledge of thetransporters utilized by specific aminosterols permits prediction of thetissues and organs these compounds will enter following administrationto an animal. In the present invention, knowledge of the tissuedistribution of the specific transporters utilized by squalamine couldguide the choice of viral infections to treat.

The net cationic charge and amphipathic character of squalamine suggestthat it could be a substrate for the principal organic cationtransporters currently identified as responsible for the pharmacokinetictrafficking of organic cations, the recently described transporters,Oct1-3 (Hayer-Zillgen, Bruss et al. 2002; Slitt, Cherrington et al.2002; Koepsell 2004; Koepsell and Endou 2004; Alnouti, Petrick et al.2006). Example 3 below demonstrates that squalamine is a substrate forthe known Oct 1-3 transporters, suggesting that squalamine has a greateropportunity for entering all tissues and organs of the body, since oneor another of these transporters in universally expressed. For example,squalamine should be capable of entering brain microvascularcapillaries, since Oct2 is known to be expressed in those cells (Sung,Yu et al. 2005). In contrast, Aminosterol 1436 is recognized solely byOct3, which is most abundantly expressed in placenta and heart, and theleast abundantly expressed of the transporters. As shown in Example 3,while squalamine readily accumulates within endothelial cells,Aminosterol 1436 does not.

Given the results of Example 4 below, showing that entry of squalamineinto the human umbilical vein endothelial cell (HUVEC) is over 2 ordersof magnitude greater than for Aminosterol 1436, reflecting thedifference in transporter affinities expressed by the endothelial cellspecific for the two aminosterols, and demonstrating that Aminosterol1436 does not appear to enter endothelial cell, it becomes apparent thatto predict whether a tissue or organ could accumulate amounts ofAminosterol 1436 required to achieve a therapeutic benefit, one wouldneed know whether Oct3 was expressed in those tissues and organs, andthe magnitude of expression of the transporter.

The discovery that squalamine is recognized by each of the known Octtransporters provides a rationale for considering the use of squalaminefor a wide variety of viruses, regardless of their particular tissue ororgan tropism.

D. Squalamine, Access to Endothelial Cells, and Viral Infections

Because it is known that endothelial cells express Oct transporters, andthat the vascular and hepatic sinusoidal endothelium are cell typestargeted by most if not all viruses that cause systemic disease inanimals, such as Hantaviruses (Geimonen, Neff et al. 2002) (Hantavirusescause two human diseases: hemorrhagic fever with renal syndrome (HFRS)and hantavirus pulmonary syndrome (HPS)), Hepatitis B virus (Breiner,Schaller et al. 2001; Rong, Huang et al. 2007), Yellow fever virus(Khaiboullina, Rizvanov et al. 2005), Dengue fever (Luplertlop and Misse2008), Varicella-Zoster (Nikkels, Debrus et al. 1995), influenza virus(Feldmann, Schafer et al. 2000; Klenk 2005; Sumikoshi, Hashimoto et al.2008; Yao, Korteweg et al. 2008), Reovirus (Verdin, King et al. 1989),Nipah Virus (Wong, Shieh et al. 2002), human rotavirus (Morrison, Gilsonet al. 2001), Parvovirus (Bultmann, Klingel et al. 2003) (e.g.,parvovirus B19 (PVB19)-associated diseases), Cytomegalovirus (Carlson,Chang et al. 2005), Vaccinia (Liu, Xu et al. 2005), Hepatitis C(Balasubramanian, Munshi et al. 2005), HIV (Bashirova, Geijtenbeek etal. 2001), Ebola (Hensley and Geisbert 2005), squalamine would beexpected to exert antiviral benefit at both the level of the tissuesthat comprise organs as well as within the vascular network of the bodyas a whole.

Furthermore, knowledge of the transporters that recognize squalaminecould provide guidance in the dosing regimens required to mosteffectively utilize squalamine as an antiviral therapeutic. For example,it is known that certain individuals who have inherited a geneticvariant of the Oct1 transporter require higher doses of metformin (adrug transported into the liver by Oct1) to maintain normal blood sugar,as compared to those who express the “wild type” transporter (Reitmanand Schadt 2007). Similarly, only Oct1 is significantly expressed inunstimulated human CD4 positive T lymphocytes, the target cell of HIV,while Oct2 expression is not observed, and Oct3 only after cytokinestimulation, suggesting that squalamine would be taken up into whiteblood cells commonly targeted by many human viruses and predictablyexert its antiviral effects in those cells (Minuesa, Purcet et al.2008).

E. Use of the Endothelial Cell Assay to Screen Active Squalamine Analogs

It has been well described in the literature that squalamine inhibitsnumerous actin-dependent processes of vertebrate endothelial cells, whenthese cells are exposed to non-cytotoxic concentrations of the molecule(Sills, Williams et al. 1998; Williams, Weitman et al. 2001) (Li,Williams et al. 2002). For squalamine to exert such an effect it mustnecessarily: (1) enter the cell, (2) achieve sufficient concentrationsto influence the dynamics of the process being measured, and (3) reducethe electrostatic potential of the inner surface of the plasma membraneto an extent the results in the displacement of proteins, such as theRhoGTPases, required for dynamic regulation of the actin cytoskeleton.

It is possible to adapt an observation reported in the literature forthe purpose of screening for derivatives of squalamine that caneffectively reduce the electrostatic potential of the plasma membrane ofa cell, a property that is required for the antiviral activity ofsqualamine. The basic screening assay involves measurement of the effectof the analog on actin stress fiber formation in endothelial cellsfollowing stimulation with VEGF or thrombin or any other stimulant knownto induce stress fiber formation in endothelial cells. In this assay,stress fiber formation can be monitored by any method that visualizestheir presence, either directly (fluorescence imaging) or indirectly(such as the measurement of the activity of enzymes or the appearance ofphosphorylated proteins, like myosin light chain kinase, and myosinlight chain, respectively).

A squalamine derivative or isomer that can effectively reduce thenegative electrostatic potential of the plasma membrane by a magnitudethat releases proteins anchored by electrostatic forces, should inhibitRho GTPase dependent processes, such as growth factor-dependent stressfiber formation in endothelial cells. Example 5, below, describes ascreening method to identify derivatives or isomers of squalamine thatexhibit comparable in vitro properties on the dynamics of the actincytoskeleton in the endothelial cell. The basic methods have beenpublished (Williams, Weitman et al. 2001). The aminosterols evaluatedwere squalamine and several derivatives and isomers (Compounds A-G), thestructures of which are shown in FIG. 4. The results, summarized inTable 3 below, showed that squalamine and the squalamine relatedcompounds A, B, and C disrupt thrombin induced stress fiber formation.This activity was not observed for squalamine analogs D, E, F, and G.

As seen in Table 4, certain stereo-isomers of squalamine, such as the3-α isomer (Compound A), the 24 S isomer (Compound B), and the 7 βhydroxy isomer (Compound C), each inhibited thrombin-induced stressfiber formation. In contrast, aminosterols D-G were inactive. CompoundD, a stereoisomer of squalamine, which differs from squalamine at asingle stereo-center (C₂₀) was inactive in the thrombin induced stressfiber formation assay. The results of Example 5 demonstrate that onlycertain isomers of squalamine can enter cells, reduce electrostaticpotential, and disturb actin cytoskeletal dynamics.

The antiviral properties of squalamine and analogs thereof disclosedherein are believed to depend upon the ability of the aminosterol toboth enter cells and also neutralize the negative electrostaticpotential of the inner surface of the plasma membrane to a degree thatcauses release of proteins anchored electrostatically to the plasmamembrane. Hence, only those compounds that can inhibit growth-factorinduced stress fiber formation as monitored in the in vitro assay above,would be expected to exhibit antiviral via the mechanism proposed forsqualamine.

F. Squalamine can Effectively Prevent Yellow Fever Infection

An example of the use of squalamine to prevent a viral infection in vivois presented in Examples 6-7 below, which demonstrate the antiviralactivity of squalamine against Yellow Fever in the hamster, a model ofYellow Fever that resembles the human infection. In FIG. 4 squalamine isadministered prior to viral infection via an intraperitoneal route,while in FIG. 5 dosing is subcutaneous. Squalamine administered at adose of 7 mg/kg/day i.p. achieved survival comparable to ribavirin,dosed optimally for this infection, at 50 mg/kg/day (FIG. 4), comparedwith placebo-treated animals that achieved a survival of 25%. In theexample illustrated in FIG. 5, a single daily dose of squalamine at 15mg/kg/day administered s.c. through day 6 post infection resulted in 70%survival, compared with 40% survival with ribavirin treatment dosed viaa single daily injection of 32 mg/kg/day over the same period, and whereplacebo treated animals achieved a survival of 15%.

Yellow fever is a member of the Flaviviridae, which includes HepatitisC, Dengue Fever Virus, Japanese Encephalitis Virus, Tick BorneEncephalitis Virus, Bovine Viral Diarrheal Virus, Classical Swine FeverVirus, Border Disease Virus, and Hepatitis G virus (Leyssen, De Clercqet al. 2000). To date only a limited number of substances have proveneffective in this model. They include antiviral nucleoside analogs suchas ribavirin and interferon-alpha (Sbrana, Xiao et al. 2004). Theexperimental model used in Example 6 has been published in detail (seee.g., Tesh, Guzman et al. 2001; Xiao, Zhang et al. 2001) and used in theevaluation of antiviral therapeutics (Sbrana, Xiao et al. 2004).

G. Squalamine can Effectively Treat a Yellow Fever Infection

An antiviral therapeutic of greatest utility should have the capacity totreat (and cure) an existing viral infection, i.e., when the individualis already suffering from the illness. With respect to Yellow Fever, noeffective therapeutic has as yet been developed for human infection. Anexample of the utility of squalamine in the treatment of an existingviral infection is presented in Example 8. To determine whethersqualamine can treat an existing Yellow Fever infection in the hamstermodel, animals were infected with a lethal inoculum of virus, and thenbegun on once daily treatment with squalamine (15 mg/kg/day, or 30mg/kg/day s.c.) beginning on day 1 or day 2 after viral administrationand continuing until day 8 and 9, respectively. Survival was monitored,and animals that remained alive by day 21 were considered “cured.” Byday 11, 100% of untreated animals had died. In contrast, of the animalsthat had received 15 mg/kg/day (from day 1-day 8) or 30 mg/kg/day (fromday 1-day 8) 60% were cured. Delay of treatment (30 mg/kg/day) until day2 and, still resulted in a cure rate of 40% (FIG. 6).

The results of this example demonstrate that squalamine can be utilizedas an effective systemic antiviral therapy in already established viralinfection. Because of the similarity in the properties shared by theflavivirus family, in addition to Yellow Fever, squalamine could be usedto treat infections caused other members of the Flaviviridae including:Dengue, Hepatitis C, West Nile, Japanese Encephalitis, Tick borneEncephalitis, St. Louis Encephalitis, Murray Valley Encephalitis,Kyasanur Fever, and any novel as yet undiscovered virus classified as amember of the Flaviviridae.

Yellow fever virus utilizes a pH dependent endosomal entry pathway toinitiate infection (Pelkmans, Helenius 2003). Based on the mechanism ofaction of squalamine disclosed in this application and the efficacy ofsqualamine in the treatment of an established infection caused by Yellowfever, squalamine could be considered for the treatment of otherinfections caused by viruses that utilize a pH dependent entry pathwaysuch as members of the Orthomyxoviridae including: Influenza A, B, C,Isavirus, Thogotovirus; members of the Rhabdomyoviridae, including:Vesiculovirus, Lyssavirus, Cytorhabdovirus, Nucleorhabdovirus,Novirhabdovirus; members of the Adenoviridae including: all HumanAdenovirus types (1-55) and species (A-G), Atadenovirus, Avidenovirus,Icthadenovirus, Mastadenovirus, Siadenovirus; members of theParvoviridae; members of the Filoviridae; members of the Iridoviridae;the Rubella virus.

H. Squalamine can Effectively Treat a Cytomegalovirus Infection (CMV)

Most viral based therapeutics are developed to inhibit a specific viraltarget that differs structurally from host cell proteins. Currentpractice generally involves the crafting of an exquisitely specific“key” to match the highly specific “lock” represented by the viraltarget; the goal is to increase specificity and minimize activityagainst host proteins, thereby reducing the toxicity of the compound. Asa consequence, most antiviral therapeutics that target the viralpathogen exhibit a narrow spectrum with respect to the viruses againstwhich the compound is active.

By virtue of its mechanism of action disclosed in this application,squalamine should exhibit a very broad spectrum of antiviral activityincluding both RNA and DNA viruses. An example of its utility in thetreatment of an infection caused by a DNA virus is presented in Example9. Herpes viruses cause many severe human diseases and remain difficultto treat effectively. In example 9 we show the utility of squalamine inthe treatment of a systemic infection by Murine cytomegalovirus (MCMV).

In Example 9 mice were inoculated via the i.p. route with a sublethalinoculum of MCMV. Squalamine was administered at a dose of 10 mg/kgdaily, beginning 1 day prior to infection and continuing daily throughday 6, by either the i.p. or s.c. routes, along with an infected cohortthat received only vehicle. On days 3, 7, and 14 animals were euthanizedand the concentration of virus present in various tissues were measured.Administration of squalamine via the i.p. route, which would have beenexpected to have resulted in the highest tissue concentrations of thecompound, was most effective, resulting in undetectable viral titers inboth liver and spleen at day 14 (FIG. 7). Dosing via the subcutaneousroute was also effective in reducing viral titers in liver and spleen,but less so than the i.p. route of administration. These results arecomparable to the best of the published responses in the mouse MCMVmodel to highly potent anti-herpes therapeutics currently on the market(Kern 2006; Ruiz, Beadle et al. 2007; Cardin, Bravo et al. 2009).

This experiment also demonstrates that squalamine is active against amember of the Herpesvirus family, and supports its use in infectionscaused by other members of the Herpes family, including Humancytomegalovirus, Herpes Simplex 1, Herpes Simplex 2, Epstein Barr Virus,Varicella Zoster Virus, Roseolovirus (HHV6 and HHV7), Kaposi's SarcomaAssociated Herpes Virus, Cercopithecine herpesvirus-1, Murinegammaherpesvirus-68, the Bovine Herpesviridae, the Canine Herpesviridae,the Equine Herpesviridae, the Feline Herpesviridae, the DuckHerpesviridae, the Chicken Herpesviridae, the Turkey Herpesviridae,Porcine Herpesviridae and any as yet undiscovered virus subsequentlyclassified as a member of the Herpesviridae.

This experiment also demonstrates, by virtue of the measured reductionin viral titers within the spleen, that squalamine administeredsystemically can effectively render virally resistant the cells of thespleen that support CMV infection, which include macrophages. Thisresult supports the use of squalamine in the treatment of all viraldiseases in which the macrophage is subject to infection.

I. Squalamine Exhibits Antiviral Activity in an Eastern EquineEncehalitis Virus Infection (EEEV), a Potential Bioterror Agent

Eastern Equine Encephalitis virus is a member of the Alphavirus familyfor which neither a vaccine nor an antiviral drug has been developed(Wang, Petrakova et al. 2007). The case fatality rate is between 30-80%for humans and up to 95% for horses and are regarded as a potentialbiodefense threat (Arrigo, Watts et al. 2008). In Example 8 we show thatsystemically administered squalamine can reduce viremia in an EEEVinfection and improve survival, in a setting where no current therapyhas any demonstrable positive effect. Golden hamsters were infected witha lethal inoculum of EEEV via the i.p. route. 1 day prior to infectionanimals received 10 mg/kg squalamine via subcutaneous dosing, whichcontinued once daily for 6 days post infection. Blood was withdrawnduring the first 4 days to monitor viral concentration. Treatment withsqualamine extended the survival of the infected animals compared tothose receiving vehicle alone (FIG. 8). Viral titers in the blood streamof squalamine treated animals were about 100-fold lower than thosereceiving vehicle, demonstrating the antviral activity of the compoundwhen administered systemically (FIG. 9). It would be anticipated thatincreasing the squalamine dose would increase the therapeutic benefit.

The results of this example demonstrate that squalamine can effectivelyreduce the concentration of virus in an animal when administeredsystemically. This experiment also demonstrates the activity ofsqualamine in treating an infection caused by a member of the Alphavirusfamily and supports its use in the treatment infection caused by othermembers of this family, including: Aura virus, Barmah Forest virus,Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equineencephalitis virus, Everglades virus, Fort Morgan virus, Getah virus,Highlands J virus Mayaro virus, Middelburg virus, Mosso das Pedras virus(78V3531, Mucambo virus, Ndumu virus, O'nyong-nyong virus, Pixuna virus,Rio Negro virus, Ross River virus, Salmon pancreas disease virus,Semliki Forest virus, Sindbis virus, Southern elephant seal virus,Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitisvirus, Western equine encephalitis virus, Whataroa virus, as well as anyas yet undiscovered virus subsequently classified as a member of theAlpaviridae.

J. Squalamine Exhibits Antiviral Activity Against Dengue Virus

Dengue is a flavivirus, responsible for the most prevalent mosquitoborne viral infection in the subtropical and tropical regions of theworld (Jessie, Fong et al. 2004). At the present time, no antiviraltherapeutic has been shown to be effective in humans. Because of thesimilarity of Dengue virus to the Yellow Fever virus it would bereasonable to assume that Dengue, like Yellow fever, would be sensitiveto the antiviral activity of squalamine.

In Example 11 the antiviral activity of squalamine is demonstratedagainst Dengue in an in vitro study involving the infection of humanmicrovascular endolthelial cells using a published procedure(Zamudio-Meza, Castillo-Alvarez et al. 2009) (FIG. 10). In thisexperiment cells were exposed to squalamine prior to addition of virusto the system. After a period of time, to permit attachment of virus tothe cells, the medium was removed and fresh medium without virus orsqualamine was replaced and the cells incubated overnight. Viralinfection was monitored immunohistochemically by the presence in thecells a viral protein (the E protein). Squalamine almost completelyinhibited viral infection at concentrations above 40 ug/ml, underconditions where cell viability did not appear to be compromised (FIG.10).

This experiment demonstrates that squalamine has direct antiviralactivity against Dengue, a non-enveloped RNA virus of the flavivirusfamily. Because of the similarity in the properties shared by theflavivirus family, in addition to Yellow Fever and Dengue, squalaminewould be expected to be active against Hepatitis C, West Nile, JapaneseEncephalitis, Tick borne Encephalitis, St. Louis Encephalitis, MurrayValley Encephalitis, Kyasanur Fever, and any novel as yet undiscoveredvirus classified as a member of the Flaviviridae.

K. Squalamine Exhibits Antiviral Activity Against Hepatitis B Virus(HBV)

Hepatitis B virus chronically infects over 350 million people worldwideand is the major cause of liver cancer worldwide. Current therapy, whichinclude interferon used alone or in combination with several nucleosideanalogues can suppress the infection in only a fraction of thepopulation and does not achieve a “cure” (Erhardt, Gobel et al. 2009).Clearly, a new agent that could interfere with Hepatitis virus growthwould be of utility. In Example 11 the antiviral activity of squalamineagainst human Hepatitis B virus is described. In this experimentHepatitis virus was introduced into a culture of primary human livercells. Squalamine was introduced either at the very start of theinfection, or after the infection had progressed for 24 hours, in eachcase squalamine being present in the system for about 16 hours. Freshmedium was replaced and the cells were maintained in culture for 12days, after which time the growth of virus was assessed. As described inExample 11, squalamine was effective in inhibiting both the early andlater stages of infection of human liver cells by Hepatitis B.

The experiment demonstrates that squalamine can exert antiviral activityagainst a human Hepatitis B virus infection of human liver. Theexperiment demonstrates that squalamine can inhibit the early phase ofinfection as well as the production of virus of cells already infected.These data support the use of squalamine for the treatment of both acuteand chronic viral hepatitis caused by Hepatitis B.

L. Squalamine Exhibits Antiviral Activity Against Hepatitis Delta Virus(HDV)

Hepatitis Delta virus is a small RNA virus that generally co-infectsindividuals already infected with Hepatitis B. About 18 million peopleare believed to be infected with this virus, for which no drug therapyexists (Abbas, Jafri et al.). In Example 13 the antiviral activity ofsqualamine against human Hepatitis Delta virus is demonstrated.Squalamine along with HDV was introduced into a culture containingprimary human hepatocytes, and exposed to the cells for about 16 hours.Fresh medium was then introduced and the cells maintained in culture for3 days after which time viral growth was measured quantitatively.Squalamine effectively inhibited the growth of Hepatitis Delta virus inhuman hepatocytes.

The experiment demonstrates that squalamine can exert antiviral activityagainst a human Hepatitis Delta virus infection of human liver. Thesedata support the use of squalamine for the treatment of acute andchronic viral hepatitis caused by Hepatitis Delta virus. Since HepatitisB and D frequently co-infect the same individual, these data wouldsupport use of squalamine for the treatment of both infectionsconcurrently.

Squalamine inhibits the replication of both Hepatitis B virus andHepatitis D virus in primary human hepatocytes, two viruses that differin their structure, mode of entry, and replicative biology, a resultanticipated by the proposed antiviral mechanism of squalamine. Theseresults strongly suggest that squalamine should be effective againstother viral infections of the human liver caused by the common Hepatitisviruses: Hepatitis A virus, Hepatitis E, Hepatitis F and Hepatitis G,and any other viral infection of the hepatocyte.

M. Squalamine Exhibits Antiviral Activity Against Human ImmunodeficiencyVirus (HIV)

Human immunodeficiency virus (HIV), including HIV-1 and HIV-2, can beeffectively treated but is not currently curable; in addition, currentHIV therapy results in the selection of drug resistant variants (Noe,Plum et al. 2005). Clearly an antiviral therapeutic that exertedantiviral activity by inducing viral resistance within the host cellwould be of benefit in a setting where viral mutation and selection ofresistant variants posed a therapeutic problem. In Example 14, theantiviral activity of squalamine against HIV is demonstrated, using apublished in vitro system (Harmon, Campbell et al.). In this ExampleHeLa cells have been engineered to express the receptors to which HIVmust attach to bind to cells and then gain entry. These cells alsocontain a reporter gene that is activated upon entry of the HIV virionand serves as the indicator of infection. In this Example squalamine atconcentrations around 20 ug/ml effectively inhibited HIV entry whileexhibiting no apparent toxicity towards the target HeLa cells.

These data support the use of squalamine for the treatment of HIV andother retroviral infections. In addition these data demonstrate thatsqualamine can block the infectivity of enveloped viruses, such as HIV,that enter cells via a pH independent fusion process. Thus, these datasupport the use of squalamine in the treatment of viral infectionscaused by viruses such as the retroviridae as well as theparamyxoviridae, including: Newcastle disease virus, Hendravirus, Nipahvirus, measles virus, Rinderpest virus, Canine distemper virus, Sendaivirus, Human parainfluenza 1, 2, 3, 4, mumps virus, Menangle virus,Tioman virus, Tuhokovirus 1, 2, 3, Human respiratory syncytial virus,avian pneumovirus, human metapneumovirus; viruses such as thepicornaviridae, including: Human enterovirus A, B, C, D, Humanrhinovirus A, B, C, Encephalomyocarditis virus, Theilovirus, Foot andmouth virus, Equine rhinitis A virus, Bovine Rhinitis B virus, HepatitisA virus, Human Parechovirus, Ljungan virus, Aichi virus, Teschovirus,Sapeloviris, Senecavirus, Tremovirus, Aviheptovirus; viruses such as therotoviridae, including: rotavirus A, B, C, D, E; viruses such as thepapovaviridae.

The discovery that squalamine and squalamine derivatives and isomers areuseful in treating and/or prevention viral infections is unexpected andsurprising, given that it is known that squalamine is inhibited fromkilling bacteria present in the blood stream, by the concentrations ofionized calcium and magnesium present in mammalian blood (supra).Squalamine exerts its antibacterial effects by directly damaging themembranes that suround the microbes. Squalamine binds to these membranesbecause they are decorated with negatively charged phospholipids thatare exposed on the outside of the microbial cell. (Note, that in thecase of animal cells, the negatively charged phospholpids are segregatedon the inner layer of the membrane, and are not exposed to theenvironment, or the blood stream). Calcium and magnesium, as cationicions, bind avidly to the negatively charged phospholipids on themicrobial surface and effectively block the sites onto which squalaminemust bind. Heretofore squalamine has been demonstrated to exert ananti-viral effect solely by directly damaging the physical integrity ofviral membranes, acting like a soap or a disinfectant, and via amechanism similar to that proposed to explain its antibacterialproperties. Based on the body of experimental data that existed prior tothe disclosure of this invention, the calcium and magnesiumconcentrations present in blood, as well as the high albumin bindingaffinity of squalamine, strongly suggested to those skilled in the artof the development of antiviral compounds that squalamine would not beexpected to be an effective in vivo treatment for viral infections.Indeed, no demonstration of squalamine's efficacy as a therapy for asystemic viral infection has been published since the report of thediscovery of the molecule in 1993, and since that date no efforts havebeen undertaken by any academic or commercial entity to develop thecompound as an antiviral therapeutic for the treatment of systemicinfections.

Moreover, to date, no published study has demonstrated the efficacy ofAminosterol 1436, a molecule closely related structurally to squalamine,in the treatment of any viral infection in an animal. Unpublishedstudies conducted at the National Institutes of Health with pig-tailedmacaques infected with SIV (simian immunodeficiency virus) failed todemonstrate any antiviral benefit from Aminosterol 1436. As describedbelow, this is likely due, in part, to failure of Aminosterol 1436 toaccess virally infected tissues, a direct consequence of the restrictedtissue expression of the specific transporter that permits 1436 to enterspecific tissues, as newly disclosed in this invention.

Another benefit of squalamine and squalamine derivatives is that viralresistance is unlikely to occur in the setting of their use.Conventional antiviral drugs, such as the protease inhibitors that areused to treat HIV, or the neuraminidase inhibitors developed to treatinfluenza, or the antiviral nucleoside analogues used to treat severaltypes of virus, target specific viral proteins and enzymes. Viruses canrapidly develop mutations within their genomes that create variants ofthe drug targets which are no longer sensitive to inhibition by thedrug. In contrast, squalamine creates an antiviral effect by inducing astate of resistance in the cells the virus is designed to target. Noknown genetic resistance mechanisms exist which a virus could acquire toovercome the inactivation of a fundamental cellular pathway required forcellular entry and infection.

III. Combination Therapy

The squalamine compositions and squalamine derivative compositions ofthe invention, may be administered alone or in combination with othertherapeutic agents. For example, the squalamine or a derivative thereofmay be administered in combination compounds including but not limitedto, chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, and/ortherapeutic treatments described below. Combinations may be administeredeither concomitantly, e.g., as an admixture, separately butsimultaneously or concurrently; or sequentially. This includespresentations in which the combined agents are administered together asa therapeutic mixture, and also procedures in which the combined agentsare administered separately but simultaneously, e.g., as throughseparate intravenous lines into the same individual. Administration “incombination” further includes the separate administration of one of thecompounds or agents given first, followed by the second.

A. Anti-Viral Agents

For example, squalamine compositions and squalamine analog compositionsof the invention with conventional antiviral therapies for treating andpreventing viral infections. For example, the squalamine and squalaminederivative compositions of the invention can be combined with any knownantiviral agent.

Designing safe and effective antiviral drugs is difficult, becauseviruses use the host's cells to replicate. This makes it difficult tofind targets for the drug that would interfere with the virus withoutharming the host organism's cells. Almost all anti-microbials, includinganti-virals, are subject to drug resistance as the pathogens mutate overtime, becoming less susceptible to the treatment. For instance, a recentstudy published in Nature Biotechnology emphasized the urgent need foraugmentation of oseltamivir (Tamiflu®) stockpiles with additionalantiviral drugs including zanamivir (Relenza®) based on an evaluation ofthe performance of these drugs in the scenario that the 2009 H1N1 ‘SwineFlu’ neuraminidase (NA) were to acquire the Tamiflu®-resistance(His274Tyr) mutation which is currently wide-spread in seasonal H1N1strains. Soundararajan et al., “Extrapolating from sequence—the 2009H1N1 ‘swine’ influenza virus”. Nature Biotechnology 27 (6) (2009). Thus,there is a need for compositions, such as those described herein, whichare useful in conjunction with conventional antiviral treatments.

Conventional antiviral treatments include, but are not limited to (1)Amantadine and rimantadine, which combat influenza and act onpenetration/uncoating; (2) Pleconaril, which works against rhinoviruses,which cause the common cold; (3) nucleotide or nucleoside analogues,such as acyclovir, zidovudine (AZT), lamivudine; (4) drugs based on“antisense” molecules, such as fomivirsen; (5) ribozyme antivirals; (6)protease inhibitors; (7) assembly inhibitors, such as Rifampicin; (8)release phase inhibitors, such as zanamivir (Relenza) and oseltamivir(Tamiflu); (9) drugs which stimulate the immune system, such asinterferons, which inhibit viral synthesis in infected cells (e.g.,interferon alpha), and synthetic antibodies (A monoclonal drug is nowbeing sold to help fight respiratory syncytial virus in babies, andantibodies purified from infected individuals are also used as atreatment for hepatitis B). Examples of antiviral drugs include, but arenot limited to, Abacavir, Aciclovir, Acyclovir, Adefovir, Amantadine,Amprenavir, Arbidol, Atazanavir, Atripla, Boceprevir, Cidofovir,Combivir, Darunavir, Delavirdine, Didanosine, Docosanol, Edoxudine,Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Entry inhibitors,Famciclovir, Fixed dose combination (antiretroviral), Fomivirsen,Fosamprenavir, Foscarnet, Fosfonet, Fusion inhibitor, Ganciclovir,Ibacitabine, Immunovir, Idoxuridine, Imiquimod, Indinavir, Inosine,Integrase inhibitor, Interferon type III, Interferon type II, Interferontype I, Interferon, Lamivudine, Lopinavir, Loviride, Maraviroc, Molixan(NOV-205), Moroxydine, Nelfinavir, Nevirapine, Nexavir, Nucleosideanalogues, Oseltamivir (Tamiflu®), Peginterferon alfa-2a, Penciclovir,Peramivir, Pleconaril, Podophyllotoxin, Protease inhibitor(pharmacology), Raltegravir, Reverse transcriptase inhibitor, Ribavirin,Rimantadine, Ritonavir, Saquinavir, Stavudine, Synergistic enhancer(antiretroviral), Tenofovir, Tenofovir disoproxil, Tipranavir,Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir (Valtrex®),Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Zalcitabine,Zanamivir (Relenza®), and Zidovudine

In certain embodiments, squalamine or a derivative thereof isadministered in combination with antiretroviral agents,nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs),non-nucleoside reverse transcriptase inhibitors (NNRTls), and/orprotease inhibitors (PIs). NRTIs that may be administered in combinationwith the squalamine or a derivative thereof, include, but are notlimited to, RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosinelddl), HIVID™(zalcitabine/ddC), ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), andCOMBIVIR™ (zidovudine/lamivudine). NNRTIs that may be administered incombination with squalamine composition, include, but are not limitedto, VIRAMUNE™ (nevirapine), RESCRIPTOR™ (delavirdine), and SUSTIVA™(efavirenz). Protease inhibitors that may be administered in combinationwith the squalamine or a derivative thereof include, but are not limitedto, CRIXIVAN™ (indinavir), NORVIR™ (ritonavir), INVIRASE™ (saquinavir),and VIRACEPT™ (nelfinavir). In a specific embodiment, antiretroviralagents, nucleoside reverse transcriptase inhibitors, non-nucleosidereverse transcriptase inhibitors, and/or protease inhibitors may be usedin any combination with squalamine or a derivative thereof to treat AIDSand/or to prevent or treat HIV infection.

Additional NRTIs include LODENOSINE™ (F-ddA; an acid-stable adenosineNRTI; Triangle/Abbott; COVIRACIL™ (emtricitabine/FTC; structurallyrelated to lamivudine (3TC) but with 3-to 10-fold greater activity invitro; Triangle/Abbott); dOTC (BCH-10652, also structurally related tolamivudine but retains activity against a substantial proportion oflanivudine-resistant isolates; Biochem Pharma); Adefovir (refusedapproval for anti-HIV therapy by FDA; Gilead Sciences); PREVEON®(Adefovir Dipivoxil, the active prodrug of adefovir; its active form isPMEA-pp); TENOFOVIR™ (bis-POC PMPA, a PMPA prodrug; Gilead); DAPD/DXG(active metabolite of DAPD; Triangle/Abbott); D-D4FC (related to 3TC,with activity against AZT/3TC-resistant virus); GW420867 (GlaxoWellcome); ZIAGEN™ (abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3′azido-2′,3′-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl(SATE)-bearing prodrug forms of beta-L-FD4C and P-L-FddC (WO 98/17281).

Additional NNRTIs include COACTINON™ (Emivirine/MKC442, potent NNRTI ofthe HEPT class; Triangle/Abbott); CAPRAVIRINE™ (AG-1549/S-1153, a nextgeneration NNRTI with activity against viruses containing the K103Nmutation; Agouron); PNU-142721 (has 20- to 50-fold greater activity thanits predecessor delavirdine and is active against K103N mutants;Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generation derivativesof efavirenz, designed to be active against viruses with the K103Nmutation; DuPont); GW420867X (has 25-fold greater activity than HBY097and is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A(naturally occurring agent from the latex tree; active against virusescontaining either or both the Y181C and K103N mutations); and Propolis(WO 99/49830).

Additional protease inhibitors include LOPINAVIR™ (ABT378/r; AbbottLaboratories); BMS-232632 (an azapeptide; Bristol-Myres Squibb);TIPRANAVIR™ (PNU-140690, a non-peptic dihydropyrone; Pharmacia &Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS232632 (an azapeptide; Bristol-Myers Squibb); L-756,423 (an indinaviranalog; Merck); DMP450 (a cyclic urea compound; Avid & DuPont); AG-1776(a peptidomimetic with in vitro activity against proteaseinhibitor-resistant viruses; Agouron); VX-175/GW433908 (phosphateprodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755 (Ciba); andAGENERASE™ (amprenavir; Glaxo Wellcome Inc.).

Additional antiretroviral agents include fusion inhibitors/gp41 binders.Fusion inhibitors/gp41 binders include T-20 (a peptide from residues643-678 of the HIV gp41 transmembrane protein ectodomain which binds togp41 in its resting state and prevents transformation to the fusogenicstate; Trimeris) and T-1249 (a second-generation fusion inhibitor;Trimeris).

Additional antiretroviral agents include fusion inhibitors/chemokinereceptor antagonists. Fusion inhibitors/chemokine receptor antagonistsinclude CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and itsanalogs, and ALX404C (a cationic peptide), T22 (an 18 amino acidpeptide; Trimeris) and the T22 analogs T134 and T140; CCR5 antagonistssuch as RANTES (9-68), AOP-RANTES, NNY-RANTES, and TAK-779; andCCR5/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Alsoincluded are CCR2B, CCR3, and CCR6 antagonists. Chemokine receptoragonists such as RANTES, SDF-1, MEP-1alpha, MIP-1beta, etc., may alsoinhibit fusion.

Additional antiretroviral agents include integrase inhibitors. Integraseinhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (adicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and relatedanthraquinones; ZINTEVIR™ (AR 177, an oligonucleotide that probably actsat cell surface rather than being a true integrase inhibitor; Arondex);and naphthols such as those disclosed in WO 98/50347.

Additional antiretroviral agents include hydroxyurea-like compounds suchas BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst);ribonucleotide reductase inhibitors such as DIDOX™ (Molecules forHealth); inosine monophosphate dehydrogenase (IMPDH) inhibitors such asVX-497 (Vertex); and mycopholic acids such as CellCept (mycophenolatemofetil; Roche).

Additional antiretroviral agents include inhibitors of viral integrase,inhibitors of viral genome nuclear translocation such as arylenebis(methylketone) compounds; inhibitors of HIV entry such as AOP-RANTES,NNY-RANTES, RANTES-IgG fusion protein, soluble complexes of RANTES andglycosaminoglycans (GAG), and AMD-3100; nucleocapsid zinc fingerinhibitors such as dithiane compounds; targets of HIV Tat and Rev; andpharmacoenhancers such as ABT-378.

Other antiretroviral therapies and adjunct therapies include cytokinesand lymphokines such as MIP-1alpha, MIP-1beta, SDF-1alpha, IL-2,PROLEUKIN™ (aldesleukin/L2-7001; Chiron), IL4, IL-10, IL-12, and IL-13;interferons such as IFN-alpha2a, IFN-alpha2b, or IFN-beta; antagonistsof TNFs, NFkappaB, GM-CSF, M-CSF, and IL-10; agents that modulate immuneactivation such as cyclosporin and prednisone; vaccines such as Remune™(HIV Immunogen), APL 400-003 (Apollon), recombinant gp120 and fragments,bivalent (B/E) recombinant envelope glycoprotein, rgp120CM235, MNrgp120, SF-2 rgp120, gp120/soluble CD4 complex, Delta JR-FL protein,branched synthetic peptide derived from discontinuous gp120 C3/C4domain, fusion-competent immunogens, and Gag, Pol, Nef, and Tatvaccines; gene-based therapies such as genetic suppressor elements(GSEs; WO 98/54366), and intrakines (genetically modified CC chemokinestargeted to the ER to block surface expression of newly synthesized CCR5(Yang et al., PNAS, 94:11567-72 (1997); Chen et al., Nat. Med.,3:1110-16 (1997)); antibodies such as the anti-CXCR4 antibody 12G5, theanti-CCR5 antibodies 2D7, 5C7, PA8, PA9, PA10, PA11, PA12, and PA14, theanti-CD4 antibodies Q4120 and RPA-T4, the anti-CCR3 antibody 7B11, theanti-gp120 antibodies 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti-Tatantibodies, anti-TNF-alpha antibodies, and monoclonal antibody 33A; arylhydrocarbon (AH) receptor agonists and antagonists such as TCDD,3,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′-tetrachlorobiphenyl, andalpha-naphthoflavone (WO 98/30213); and antioxidants such asgamma-L-glutamyl-L-cysteine ethyl ester (gamma-GCE; WO 99/56764).

B. Anti-Inflammatory Agents

In certain embodiments, the squalamine or a derivative thereof isadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the squalamine ora derivative thereof include, but are not limited to, corticosteroids(e.g. betamethasone, budesonide, cortisone, dexamethasone,hydrocortisone, methylprednisolone, prednisolone, prednisone, andtriamcinolone), nonsteroidal anti-inflammatory drugs (e.g., diclofenac,diflunisal, etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen,indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam,nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac,tenoxicam, tiaprofenic acid, and tolmetin), as well as antihistamines,aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acidderivatives, pyrazoles, pyrazolones, salicylic acid derivatives,thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone,nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime,proquazone, proxazole, and tenidap.

IV. Formulations

Compositions for pharmaceutical use typically comprise apharmaceutically acceptable carrier, for example, solvents, dispersionmedia, coatings, isotonic and absorption delaying agents and the like,and combinations comprising one or more of the foregoing carriers asdescribed, for instance, in REMINGTON'S PHARMACEUTICAL SCIENCES, 15thEd. Easton: Mack Publishing Co. pp. 1405-1412 and 1461-1487 (1975), andTHE NATIONAL FORMULARY XIV 14th Ed., Washington: American PharmaceuticalAssociation (1975). Suitable carriers include, but are not limited to,calcium carbonate, carboxymethylcellulose, cellulose, citric acid,dextrate, dextrose, ethyl alcohol, glucose, hydroxymethylcellulose,lactose, magnesium stearate, maltodextrin, mannitol, microcrystallinecellulose, oleate, polyethylene glycols, potassium diphosphate,potassium phosphate, saccharose, sodium diphosphate, sodium phosphate,sorbitol, starch, stearic acid and its salts, sucrose, talc, vegetableoils, water, and combinations comprising one or more of the foregoingcarriers. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the compositions of the presentinvention, their use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

A. Pharmaceutical Carriers

While it is possible for a squalamine or a derivative thereof to beadministered alone, it is preferable to present it as a pharmaceuticalformulation, together with one or more acceptable carriers. Thecarrier(s) must be “acceptable” in the sense of being compatible withthe squalamine or a derivative thereof and not deleterious to therecipients thereof. Typically, the carriers will be water or salinewhich will be sterile and pyrogen free. Squalamine or a derivativethereof is particularly well suited to formulation in aqueous carrierssuch as sterile pyrogen free water, saline or other isotonic solutionsbecause of their extended shelf-life in solution. For instance,pharmaceutical compositions of the invention may be formulated well inadvance in aqueous form, for instance, weeks or months or longer timeperiods before being dispensed.

Generally, the formulations are prepared by contacting the squalamine ora derivative thereof uniformly and intimately with liquid carriers orfinely divided solid carriers or both. Then, if necessary, the productis shaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably comprises minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such asgelatin, serum albumin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

In instances where aerosol administration is appropriate, the squalamineor a derivative thereof can be formulated as aerosols using standardprocedures. The term “aerosol” includes any gas-borne suspended phase ofa squalamine or a derivative thereof which is capable of being inhaledinto the bronchioles or nasal passages, and includes dry powder andaqueous aerosol, and pulmonary and nasal aerosols. Specifically, aerosolincludes a gas-bome suspension of droplets of squalamine or a derivativethereof, as may be produced in a metered dose inhaler or nebulizer, orin a mist sprayer. Aerosol also includes a dry powder composition of acompound of the invention suspended in air or other carrier gas, whichmay be delivered by insufflation from an inhaler device, for example.See Ganderton & Jones, Drug Delivery to the Respiratory Tract (EllisHorwood, 1987); Gonda, Critical Reviews in therapeutic Drug CarrierSystems, 6:273-313 (1990); and Raeburn et al., Pharmacol. Toxicol.Methods, 27:143-159 (1992).

B. Exemplary Dosage Forms

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Any pharmaceutically acceptable dosage form may be employed in themethods of the invention. A preferred dosage form is an orallyadministered dosage form, such as a tablet or capsule. Such methodsinclude the step of bringing into association the squalamine or aderivative thereof with the carrier that constitutes one or moreaccessory ingredients. In general the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both, and then,if necessary, shaping the product.

1. Exemplary Dosage Forms

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulationappropriate for the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampules, vials or syringes, and may bestored in a freeze-dried (Lyophilised) condition requiring only theaddition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders.

Formulations or compositions of the invention may be packaged togetherwith, or included in a kit with, instructions or a package insert. Forinstance, such instructions or package inserts may address recommendedstorage conditions, such as time, temperature and light, taking intoaccount the shelf-life of the squalamine or a derivative thereof. Suchinstructions or package inserts may also address the particularadvantages of the squalamine or a derivative thereof, such as the easeof storage for formulations that may require use in the field, outsideof controlled hospital, clinic or office conditions.

The squalamine or a derivative thereof can also be included innutraceuticals. For instance, squalamine or a derivative thereof may beadministered in natural products, including milk or milk productobtained from a transgenic mammal which expresses alpha-fetoproteinfusion protein. Such compositions can also include plant or plantproducts obtained from a transgenic plant which expresses the squalamineor a derivative thereof. The squalamine or a derivative thereof can alsobe provided in powder or tablet form, with or without other knownadditives, carriers, fillers and diluents. Exemplary nutraceuticals aredescribed in Scott Hegenhart, Food Product Design, December 1993.

The invention also provides methods of treatment and/or prevention ofdiseases or disorders (such as, for example, any one or more of thediseases or disorders disclosed herein) by administration to a subjectof an effective amount of a squalamine or a derivative thereof in apharmaceutically acceptable carrier.

The squalamine or a derivative thereof will be formulated and dosed in afashion consistent with good medical practice, taking into account theclinical condition of the individual patient (especially the sideeffects of treatment with the squalamine or a derivative thereof alone),the site of delivery, the method of administration, the scheduling ofadministration, and other factors known to practitioners. The “effectiveamount” for purposes herein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofthe squalamine or a derivative thereof administered parenterally perdose will be in the range of about 0.1 mg/kg/day to 20 mg/kg/day ofpatient body weight, although, as noted above, this will be subject totherapeutic discretion. An intravenous bag solution may also beemployed. The length of treatment needed to observe changes and theinterval following treatment for responses to occur appears to varydepending on the desired effect.

“Pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous, transdermal,and intraarticular injection and infusion.

Squalamine or a derivative thereof is also suitably administered bysustained-release systems. Examples of sustained-release squalamine or aderivative thereof compositions are administered orally, rectally,parenterally, intracistemally, intravaginally, intraperitoneally,topically (as by powders, ointments, gels, drops or transdermal patch),bucally, or as an oral or nasal spray. “Pharmaceutically acceptablecarrier” refers to a non-toxic solid, semisolid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.The term “parenteral” as used herein refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion. Additionalexamples of sustained-release squalamine or a derivative thereof includesuitable polymeric materials (such as, for example, semi-permeablepolymer matrices in the form of shaped articles, e.g., films, ormirocapsules), suitable hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)),poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res., 15:167-277 (1981), and Langer, Chem. Tech., 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Sustained-release squalamine or a derivative thereof also includeliposomally entrapped squalamine or a derivative thereof (see generally,Langer, Science, 249:1527-1533 (1990); Treat et al., in Liposomes in theTherapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler(eds.), pp. 317-327 and 353-365 (Liss, N.Y., 1989). Liposomes comprisingthe squalamine or a derivative thereof are prepared by methods known perse: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA),82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA),77:40304034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. percent cholesterol, the selected proportionbeing adjusted for the optimal therapeutic.

In yet an additional embodiment, the squalamine or a derivative thereofare delivered by way of a pump (see Langer, supra; Sefton, CRC Crit.Ref. Biomed. Eng., 14:201 (1987); Buchwald et al., Surgery, 88:507(1980); Saudek et al., N. Engl. J. Med., 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the squalamine or aderivative thereof is formulated generally by mixing it at the desireddegree of purity, in a unit dosage injectable form (solution,suspension, or emulsion), with a pharmaceutically acceptable carrier,i.e., one that is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation. For example, the formulation preferably does not includeoxidizing agents and other compounds that are known to be deleterious tothe therapeutic.

Any pharmaceutical used for therapeutic administration can be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Squalamine or aderivative thereof generally is placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

Squalamine or a derivative thereof ordinarily will be stored in unit ormulti-dose containers, for example, sealed ampoules or vials, as anaqueous solution or as a lyophilized formulation for reconstitution. Asan example of a lyophilized formulation, 10-ml vials are filled with 5ml of sterile-filtered 1% (w/v) aqueous squalamine or a derivativethereof solution, and the resulting mixture is lyophilized. The infusionsolution is prepared by reconstituting the lyophilized squalamine or aderivative thereof using bacteriostatic Water-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thesqualamine or a derivative thereof. Associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,use or sale for human administration. In addition, the squalamine or aderivative thereof may be employed in conjunction with other therapeuticcompounds.

2. Dosages

Examples of dosages of squalamine tolerated by humans are well known inthe art. For example, Hao et al., (2003). “A Phase I and pharmacokineticstudy of squalamine, an aminosterol angiogenesis inhibitor.” Clin CancerRes 9(7): 2465-71, describes exemplary dosages for a 5-day continuousi.v. infusion every 3 weeks for treating advanced solid malignancies.Dose levels ranging from 6 to 700 mg/m(2)/day. Hepatotoxicity,characterized by brief, asymptomatic elevations in transaminases andhyperbilirubinemia, was the principal dose-limiting toxicity ofsqualamine. At 700 mg/m(2)/day, two of three patients developed grade 4hyperbilirubinemia, which precluded further dose escalation. At 500mg/m(2)/day, one of seven patients experienced dose-limiting grade 4hyperbilirubinemia and grade 3 neurosensory changes, which resolved soonafter treatment. Squalamine pharmacokinetics were dose-proportional. At500 mg/m(2)/day, the mean (percentage coefficient of variation)clearance, half-life, and volume of distribution of squalamine were 2.67liters/h/m(2) (85%), 9.46 h (81%), and 36.84 liters/m(2) (124%),respectively, and steady-state concentrations [20.08 micro g/ml (13%)]were well above those that inhibit angiogenesis in preclinical models.The study concluded that at a dose of 500 mg/m(2)/day, squalamine iswell tolerated.

In addition, Herbst et al., (2003). “A phase I/IIA trial of continuousfive-day infusion of squalamine lactate (MSI-1256F) plus carboplatin andpaclitaxel in patients with advanced non-small cell lung cancer.” ClinCancer Res 9(11): 4108-15, also describes exemplary therapeutic dosageof squalamine. This reference describes a Phase I/IIA study designed toassess the safety, clinical response, and pharmacokinetics of squalaminewhen administered as a 5-day continuous infusion in conjunction withstandard chemotherapy every 3 weeks in patients with stage IIIB (pleuraleffusion) or stage IV non-small cell lung cancer. Patients withchemotherapy-naive non-small cell lung cancer were treated withescalating doses of squalamine in combination with standard doses ofpaclitaxel and carboplatin. Paclitaxel and carboplatin were administeredon day 1, followed by squalamine as a continuous infusion on days 1-5,every 21 days. The starting dose of squalamine was 100 mg/m(2)/day andescalated to 400 mg/m(2)/day; two of three patients at 400 mg/m(2)/dayhad dose-limiting toxicity that included grade 3/4 arthralgia, myalgia,and neutropenia. On the basis of safety and toxicity, 300 mg/m(2)/daywas selected as the Phase II dose of squalamine in this combinationregimen. The combination of squalamine given continuously daily for 5days, with paclitaxel and carboplatin given on day 1, was welltolerated.

C. Adjuvants

The squalamine or a derivative thereof may be administered alone or incombination with adjuvants. An adjuvant is a substance that indirectlyenhances the therapeutic activity of squalamine by stimulating theantiviral arm of the innate and/or the adaptive immune system. Adjuvantsthat may be administered with the squalamine or a derivative thereofinclude, but are not limited to, cytokines and/or interleukins (such asIL2, IL3, IL4, IL5, IL6, IL7, IL8, IL-9, IL10, IL-11, IL12, IL13, IL-14,IL15, IL16, IL-17, IL-18, IL-19, IL-20, IL-21, anti-CD40, CD40L,IFN-gamma, TNF-alpha, IL-1alpha, IL-1beta), Lipid A, includingmonophosphoryl lipid A, bacterial products, endotoxins, cholesterol,fatty acids, aliphatic amines, paraffinic and vegetable oils, threonylderivative, and muramyl dipeptide, alum, alum plus deoxycholate(ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG (e.g.,THERACYS®), MPL and nonviable preparations of Corynebacterium parvum. Ina specific embodiment, squalamine or a derivative thereof isadministered in combination with alum. In another specific embodiment,squalamine or a derivative thereof is administered in combination withQS-21. Further adjuvants that may be administered with the squalamine ora derivative thereof include, but are not limited to, Monophosphoryllipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminumsalts, MF-59, and Virosomal adjuvant technology.

D. Vaccines

Vaccines that may be administered with the squalamine or a derivativethereof include any antigen capable of eliciting an immune response. Thevaccine may be comprised of either live or inactivated virus. Exemplaryvaccines include, but are not limited to, vaccines directed towardprotection against MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, Haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, pertussis, PA-toxin (e.g., anthrax), HumanImmunodeficiency Virus (HIV-1 and HIV-2), Avian Flu antigen (e.g., H5N1;avian influenza virus A/FPV/Rostock/34 (H7N1) (FPV)), cancer, SevereAcute Respiratory Syndrome (SARS), and tuberculosis. Useful antigensinclude but are not limited to viral, prion, bacterial, parasitic,mycotic, etc. antigens.

Combinations may be administered either concomitantly, e.g., as anadmixture, separately but simultaneously or concurrently; orsequentially. In addition, as used herein “combination administration”includes compounds which are attached to the squalamine or a derivativethereof. This also includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

V. Methods of treating and/or preventing viral Infections usingsqualamine compositions

The squalamine compositions and squalamine derivative compositions areparticularly useful in decreasing the infectivity, morbidity, and/orrate of mortality associated with a variety of viruses.

The squalamine compositions described herein may be administered by anyconventional method including parenteral (e.g. subcutaneous orintramuscular) injection or intravenous infusion, orally, rectally,parenterally, intracistemally, intravaginally, intraperitoneally,topically (as by powders, ointments, gels, drops or transdermal patch),otically, ocularly, bucally, pulmonarily (e.g., as an oral or nasalspray or as an aerosol dispersion). The treatment may consist of asingle dose or a plurality of doses over a period of time. For example,in preferred embodiments the squalamine or squalamine derivative isadministered parenterally (e.g. subcutaneously injection orintramuscularly injection) or intravenous infusion.

Squalamine has been administered intravenously through peripheral orcentral veins. Squalamine is widely distributed throughout all tissuesincluding the brain. Squalamine exhibits many of the metabolic/clearancecharacteristics of a bile salt. When administered orally squalamine isalmost fully captured by the liver and likely enters an enterohepaticcycle during its subsequent metabolism, suggesting that appropriatehepatic diseases could be treated by oral administration.

The methods and compositions may be formulated into a single or separatepharmaceutically acceptable syringeable composition. In this case, thecontainer means may itself be an inhalant, syringe, pipette, eyedropper, or other like apparatus, from which the formulation may beapplied to an infected area of the body, such as the lungs, injectedinto an animal, or even applied to and mixed with the other componentsof the kit.

The methods and compositions, or components of the methods andcompositions can be formulated in a single formulation, or can beseparated into binary formulations for later mixing during use, as maybe desired for a particular application. Such components canadvantageously be placed in kits for use against microbial infections,decontaminating instruments and the like. Such kits may contain all ofthe essential materials and reagents required for the delivery of theformulations to the site of their intended action as well as any desiredinstructions.

A. Viral Diseases

Squalamine or a derivative thereof can be used to treat or detect anyknown viral infectious agent, including but not limited to any viralagent described herein. Viruses are one example of an infectious agentthat can cause disease or symptoms that can be treated or detected bysqualamine or a derivative thereof. Examples of viruses, include, butare not limited to the following DNA and RNA viruses and viral families:“African Swine Fever Viruses,” Arbovirus, Adenoviridae, Arenaviridae,Arterivirus, Astroviridae, Baculoviridae, Bimaviridae, Birnaviridae,Bunyaviridae, Caliciviridae, Caulimoviridae, Circoviridae,Coronaviridae, Cystoviridae, Dengue, EBV, HIV, Deltaviridae, Filviridae,Filoviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae(such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Iridoviridae,Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae),Myoviridae, Orthomyxoviridae (e.g., Influenza A, Influenza B, andparainfluenza), Papiloma virus, Papovaviridae, Paramyxoviridae, Prions,Parvoviridae, Phycodnaviridae, Picornaviridae (e.g. Rhinovirus,Poliovirus), Poxviridae (such as Smallpox or Vaccinia), Potyviridae,Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,Lentivirus), Rhabdoviridae, Tectiviridae, and Togaviridae (e.g.,Rubivirus). In one embodiment, the virus is herpes, pox, papilloma,corona, influenza, hepatitis, sendai, sindbis and vaccinia viruses, westnile, hanta, or viruses which cause the common cold. Viruses fallingwithin these families can cause a variety of diseases or symptoms,including, but not limited to: arthritis, bronchiollitis, respiratorysyncytial virus, encephalitis, eye infections (e.g., conjunctivitis,keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, ChronicActive, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valleyfever, yellow fever, meningitis, opportunistic infections (e.g., AIDS),pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles,Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia.

Squalamine or a derivative thereof can be used to treat or detect any ofthese symptoms or diseases. In specific embodiments, squalamine or aderivative thereof is used to treat viral meningitis, Dengue, EBV,and/or hepatitis (e.g., hepatitis B). In an additional specificembodiment squalamine or a derivative thereof is used to treat patientsnonresponsive to one or more other commercially available hepatitistherapies. In a further specific embodiment, squalamine or a derivativethereof is used to treat AIDS.

In another embodiment, squalamine or a derivative thereof is used totreat a chronic disease suspected to be of viral origin. Theseconditions include diseases such as multiple sclerosis, Type I and TypeII diabetes, atherosclerosis, cardiomyopathies, Kawaski disease,aplastic anemia, and so on.

EXAMPLES

The following examples are provided to illustrate the present invention.It should be understood, however, that the invention is not to belimited to the specific conditions or details described in theseexamples. Throughout the specification, any and all references to apublicly available document, including a U.S. patent, are specificallyincorporated by reference.

Example 1

This example demonstrates the capacity of squalamine to displaceproteins associated with the inner surface of the plasma membranethrough electrostatic interactions.

The basic methods utilized in this example are described in thepublished literature (Yeung, Terebiznik et al. 2006; Yeung, Gilbert etal. 2008). The methodology described in these published reports providetools to measure the electrostatic potential of the inner surface of thecell membrane, that being the side faces the cytoplasm of a cell. Thepublished method comprises introducing into a cell a fluorescent proteinthat contains a strong cationic (positively charged) region. Thisfluorescent protein, because of its strong positive charge will bind tonegatively charged membranes, causing the membrane to “light up” as aconsequence of the attachment to it of the fluorescent probe. Should thestrong negative charge on the plasma membrane be lost, for example, dueto hydrolysis of the phosphatidyl serine, the fluorescent probe wouldfall off and diffuse into the cytoplasm. Thus the segregation of thefluorescent signal on the membrane indicates the persistence of theavailable negative charge of the cytoplasmic face of the membrane. Ingeneral, these probes are comprised of a standard fluorescent protein(like green fluorescent protein) to which a short peptide is fusedcorresponding to the cationic region of proteins that anchor to thecytoplasmic face of the plasma membrane. The tools and methods describedin the literature and used in this example have demonstrated thatelectrostatic interactions between the negatively charged inner surfaceof the cytoplasmic surface of the plasma membrane and the cationicregions anchor many members of the Ras/Rho small GTPases to theirmembrane locus and confirm that the anionic phospholipids on the innerface of the plasma membrane create a strong, negatively charged surface.(Yeung, Terebiznik et al. 2006; Yeung, Gilbert et al. 2008).

A RAW 264.7 macrophage line was transfected with engineered recombinantvectors to generate cells that expressed two peptide probes, each linkedto a red or green fluorescent protein to permit microscopicvisualization. “Trunc Cat Tail” (GFP-ARDGRRRRRRARARCVIM) is a highlycationic probe. “H-Ras” (RFP—full length H-Ras) is a member of the Rasfamily of proteins that associates with the plasma membrane principallythrough interactions dominated by two fatty acid chains covalentlyattached to the carboxyterminal end of the molecule. This bindinginteraction depends principally on hydrophobic interactions (“oilprefers to mix with oil over water”) rather than through electrostatic(charge based) interactions. Prior to exposure of these cells tosqualamine, both H-Ras and Trunc Cat Tail can be seen associated withthe plasma membrane (FIG. 2, upper set of panels). Following theaddition of squalamine (10 micromolar) to the culture medium in whichthe cells are bathed, the Truc Cat Tail probe is displaced into thecytoplasm, while the H-Ras probe remains associated with the membrane.

Results: Since the H-Ras probe has not been displaced, it can beconcluded that squalamine did not disrupt the physical integrity of themembrane, and cause widespread detachment of all associated proteins.The release of the Trunc Cat Tail probe strongly suggests thatsqualamine has reduced the net negative electrostatic potential,resulting in release of the cationic probe from the plasma membrane, itsentry into the cytoplasm, and relocation to favorable anchoring sites onvarious intracellular membranes.

Example 2

In this experiment, the results of which have been published (Yeung,Gilbert et al. 2008), the RAW264.7 cell line has been engineered toexpress a red tagged fluorescent protein (“Lact-C2”) that binds avidlyto phosphatidylserine through forces that are not based solely onelectrostatic potential. In addition the cell also expresses a greentagged fluorescent cationic fragment (“R-pre”), similar in sequence anddesign to the “Trunc Cat Tail” probe discussed above.

As seen in FIG. 3, before addition of squalamine, both probes are seento be associated with the plasma membrane, as expected. In addition toits localization at the plasma membrane, the Lact-C2 probe can be seendecorating intracellular membranes as well, reflecting the sites withinthe cell that are rich in phosphatidylserine (Yeung, Gilbert et al.2008). The restricted localization of R-pre to the plasma membranereflects the highly cationic charge on R-pre (8 positive charges) andthe fact that the cytoplasmic surface of the plasma membrane exhibitsthe strongest negative electrostatic potential of any major membrane inan animal cell (Yeung, Gilbert et al. 2008).

Within several minutes following exposure of these cells to squalamine(80 μM, 30 minutes), R-pre was displaced from its residence on theplasma membrane to other areas within the cell's interior. In contrast,exposure of these cells to squalamine did not alter the localization ofLact-C2.

This example demonstrates that squalamine reduces the electrostaticpotential of the membrane into which it sits, displacing proteins boundvia “non-specific” electrostatic forces, but cannot necessarily displacea protein that binds to phosphatidylserine through forces based onchemically specific features of that lipid.

Example 3

This example determines whether squalamine is a substrate for theorganic cation transporter (Oct). This determination employed a standardtransport competition assay, described in the literature (Lips, Volk etal. 2005).

Chinese hamster ovary cells, previously engineered to over-express aspecific human Oct transporter were used in the assay. In this example,the effect of a specific concentration of an aminosterol is determinedon the intracellular uptake of radiolabelled 1-methyl-4-phenylpyridine(MPP) (at 0.01 μM), measured after a 1-second exposure. The extent towhich the added compound inhibits the uptake is recorded (as %inhibition). IC50 refers to the concentration of squalamine or 1436required to inhibit MPP uptake by 50%. Table 1 below describes theaffinity of squalamine and Aminosterol 1436 for human organic cationtransporters (Human Oct 1, Human Oct 2, and Human Oct 3) based upon theMPP uptake inhibition assay.

TABLE 1 Affinity of Squalamine and 1436 for the human organic cationtransporters based on an MPP uptake inhibition assay % Inhibition of MPP% Inhibition of MPP uptake caused by 100 μM uptake caused by 100 μMTransporter Squalamine (IC50) 1436 (IC50) Human Oct 1 25% (34 μM) 0 (upto 1 mM 1436) Human Oct 2 40% (50 μM) 0 (up to 1 mM 1436) Human Oct 360% (83 μM, 16 μM) 70% (29 μM, 34 μM)

The values obtained for squalamine and Aminosterol 1436 compare inmagnitude with substances previously described in the literature andalready known to be transported via Oct proteins (Koepsell 2004).

Thus, the results of this example demonstrate that squalamine is asubstrate for the known Oct1 3 transporters, suggesting that squalaminehas a greater opportunity for entering all tissues and organs of thebody, since one or another of these transporters in universallyexpressed. For example, squalamine should be capable of entering brainmicrovascular capillaries, since Oct 2 is known to be expressed in thosecells (Sung, Yu et al. 2005). In contrast, Aminosterol 1436 isrecognized solely by Oct 3, which is most abundantly expressed inplacenta and heart, and the least abundantly expressed of thetransporters. Thus, this Example predicts that squalamine should readilyaccumulate within endothelial cells, while Aminosterol 1436 should not,which is indeed the case (as shown in Example 4).

Example 4

The purpose of this example was to determine the kinetics of squalamineand Aminosterol 1436 update into endothelial cells.

Human umbilical endothelial cells were grown as described (Sills,Williams et al. 1998). Flasks containing cells at 75% confluence wereincubated with ³H squalamine or ³H-1436 (2 uCi/ml, 1 μg/ml) in 5 mlfresh medium. After various times the medium was removed, washed withPBS and cells released by scrapping. The cells were counted, and thencollected by brief centrifugation, followed by resuspension in 1 ml ofbuffer containing 10 mM Tris-HCl, 2 mM CaCl₂, 1 mM Mg Cl₂, 1 mM DTT, andlysing with 10 strokes of a loose fitting Dounce homogenizer.Radioactivity contained in aliquots of the homogenates was measured byscintillation counting. The results are shown in Table 2, below.

TABLE 2 Kinetics of uptake of squalamine and Aminosterol 1436 intoendothelial cells Molecules/cell (×10⁻⁶) Time (min) SqualamineAminosterol 1436 5 250 2 30 400 60 500 3 120 500 180 600 2

This example demonstrates that the entry of squalamine into the HUVEC isover 2 orders of magnitude greater than for Aminosterol 1436, reflectingthe difference in transporter affinities expressed by the endothelialcell specific for the two aminosterols. Aminosterol 1436 cannot enterthe endothelial cell because this cell type does not express Oct 3, thesole transporter currently known to permit Aminosterol 1436 to gainentry into the membrane; squalamine, in contrast, can readily enterthrough Oct 1 and Oct 2, both of which are known to be expressed inendothelial cells.

As reported below, Aminosterol 1436 does not appear to functionallyneutralize the electrostatic potential of the plasma membrane of theendothelial cell, best understood as a consequence of the failure of thecompound to enter this cell type.

Example 5

This example is directed to using a screening method to identify analogsof squalamine that exhibit in vitro properties comparable to squalamineon the dynamics of the actin cytoskeleton in the endothelial cell.

The basic methods have been published (Williams, Weitman et al. 2001).HUVECs were grown on fibronectin-covered coverslips in EGM medium. Thecells were then incubated in the presence of thrombin (20 ng/ml), andeither the aminosterol (20 μM), or an equivalent volume of water, for 2hours. The treated cells were fixed with 3.7% formaldehyde,permeabilized with 0.1% Triton X-100, and incubated with a 1:100dilution of murine anti-VE-cadherin monoclonal antibody. Following arinse, the cells were exposed to a 1:400 dilution of an FITC-goatantimurine antibody and 250 ng/ml TRITC phalloidin. The stainedcoverslips were visualized by fluorescence microscopy.

The aminosterols evaluated were squalamine and several analogs, thestructures of which are presented in Table 3. The results of the assayare summarized in Table 4.

TABLE 3

Compound Structure Squalamine X = 3β-H₂N—(CH₂)₄—NH—(CH₂)₃—NH—, Z =7α-OH, Y = 20R—CH₃, W = 24R-sulfate A X = 3β-H₂N—(CH₂)₄—NH—(CH₂)₃—NH—, Z= 7β-OH, Y = 20R—CH₃, W = 24R-sulfate B X = 3α-H₂N—(CH₂)₄—NH—(CH₂)₃—NH—,Z = 7α-OH, Y = 20R—CH₃, W = 24R-sulfate C X =3β-H₂N—(CH₂)₄—NH—(CH₂)₃—NH—, Z = 7α-OH, Y = 20R—CH₃, W = 24S-sulfate D X= 3β-H₂N—(CH₂)₄—NH—(CH₂)₃—NH—, Z = 7α-OH,, Y = 20S—CH₃, W = 24R-sulfateE X = 3-NH3, Z = 7α-OH, Y = 20R—CH₃, W = 24R-sulfate F X =3β-H₂N—(CH₂)₃—O—(CH₂)₃—NH—, Z = 7α-OH , Y = 20R—CH₃, W = 24R-sulfate G X= 3β-spermine, Z = 7α-OH, Y = 20R—CH₃, (Aminosterol 1436) W =24R-sulfate

TABLE 4 Disruption of thrombin induced stress fiber Aminosterolformation Squalamine Yes A Yes B Yes C Yes D No E No F No G No

As seen in Table 4, certain stereo-isomers of squalamine, such as the3-α isomer, the 24 S isomer, and the 7 β hydroxy isomer, each inhibitedthrombin-induced stress fiber formation. In contrast, aminosterols D-Gwere inactive. Compound D, a stereoisomer of squalamine, differs fromsqualamine at a single stereo-center (C₂₀). Compound E, differs fromsqualamine by having a single amino group fixed to 3C instead of aspermidine; Compound F differs from squalamine by having aspermidine-like moiety that carries only 2 positive charges instead ofthe 3 carried by spermidine; and G is Aminosterol 1436, identical tosqualamine, expect for the presence of a spermine moiety (4 charges) inplace of a spermidine (3 charges) at C3.

The results of this Example demonstrate that only certain isomers ofsqualamine can enter cells, reduce electrostatic potential, and disturbactin cytoskeletal dynamics. These data highlight the highly specificstructural constraints that must be met by active squalamine analogues,and must reflect the highly specific nature of the molecularinteractions responsible for the biological effect being observed.

The antiviral properties of squalamine disclosed herein are believed todepend on the ability of the aminosterol to both enter cells and alsoneutralize the negative electrostatic potential of the inner surface ofthe plasma membrane to a degree that causes release of proteins anchoredelectrostatically to the plasma membrane. Hence, only those compoundsthat can inhibit growth-factor induced stress fiber formation asmonitored in the in vitro assay above, would be expected to exhibitantiviral via the mechanism proposed for squalamine.

Example 6

The purpose of this example was to demonstrate the effectiveness ofsqualamine in treating a viral infection, such as Yellow Fever. Theresults show that squalamine can effectively prevent Yellow Feverinfection in inoculated hamsters.

The hamster is used as an animal model to study yellow fever because itdevelops a disease following infection with the Yellow fever virus thatresembles the human Yellow Fever infection. Yellow fever is a member ofthe Flaviviridae, which includes Hepatitis C, Dengue Fever Virus,Japanese Encephalitis Virus, Tick Borne Encephalitis Virus, Bovine ViralDiarrheal Virus, Classical Swine Fever Virus, Border Disease Virus, andHepatitis G virus (Leyssen, De Clercq et al. 2000). To date, only alimited number of substances have proven effective in this model. Theyinclude antiviral nucleoside analogs such as ribavirin andinterferon-alpha (Sbrana, Xiao et al. 2004).

The experimental model used in this example has been published in detail(Tesh, Guzman et al. 2001; Xiao, Zhang et al. 2001) and used in theevaluation of antiviral therapeutics ((Sbrana, Xiao et al. 2004).

Study Design: The study design for this Example is given in Table 5,below.

TABLE 5 Study Design Parameter Virus Jimenez hamster-adapted yellowfever virus inoculated intraperitoneal (i.p.) Number of animals 10/group20/placebo group Treatment route intraperitoneal (i.p.) Start ofTreatment −24 hours Treatment times daily for 8 days (−24 h to 6 dayspost- virus inoculation (dpi)) Parameters Percent survivors, serumALT/AST levels determined on day 6, mean day to death, weight changebetween 3 and 6 dpi Infected Groups Group 1- Squalamine 7.7 mg/kg/d, qdGroup 3- Squalamine 2.5 mg/kg/d, qd Group 5- Squalamine 0.7 mg/kg/d, qdGroup 7- Ribavirin 50 mg/kg/d, bid Group 9- Saline

Results: As seen in FIG. 4, survival increased in response to increasingdoses of squalamine. At 7.7 mg/kg, administered as a single daily dose,the survival fraction of the squalamine treated group was nearlyidentical to the group that received ribavirin (50 mg/kg/day). (Note:“bid” in Table 5 refers to twice a day dosing, while “qd” refers to oncea day dosing.)

These results demonstrate the effectiveness of squalamine as anantiviral agent.

Example 7

The purpose of this example was to evaluate the effectiveness ofsqualamine in treating a viral infection, such as Yellow Fever, whenadministered subcutaneously, in a head-to-head comparison withribavirin, over a comparable dosing schedule. Ribavirin is a nucleosideanalogue that is effective in the hamster model when administered via anoptimized dosing schedule. Ribavirin is used as an antiviral therapeuticin humans, mainly for the treatment of Hepatitis C, in conjunction withInterferon-α.

Experimental design: Hamsters were randomly assigned to groups, with 10included in each and 20 placebo-treated controls. A 10⁻⁴ dilution(10^(2.0) CCID₅₀/ml) of the virus was prepared in minimal essentialmedia. Hamsters were injected intraperitoneally (i.p.) with 0.1 ml ofthe diluted virus (10 CCID₅₀/animal). Squalamine was administratedsubcutaneously (s.c.) at a total daily dose 15 mg/kg/d, given once a day(qd) beginning 24 h before introduction of virus and ending on 6 dayspost infection (dpi). Ribavirin was administered intraperitonealy (i.p.)at doses of 3.2, 10, or 32 mg/kg/d administered once daily beginning 24h before introduction of virus and ending on 6 days post infection(dpi). Mortality was observed daily for 21 days, and weight was recordedon 0, 3, and 6 dpi.

Statistical analysis: Survival data were analyzed using the Wilcoxonlog-rank survival analysis and all other statistical analyses were doneusing one-way ANOVA using a Bonferroni group comparison (Prism 5,GraphPad Software, Inc).

Results: Treatment with squalamine resulted in 70% survival comparedwith 15% of animals receiving vehicle alone (FIG. 5). About 40% of theanimals receiving ribavirin at the highest dose (32 mg/kg/day) survived,while those receiving lower doses fared no better than theplacebo-treated cohort.

These results illustrate the effectiveness of squalamine as an antiviralagent and demonstrate that its antiviral activity in an animal iscomparable to a well-studied antiviral agent currently in use as a humantherapeutic drug against another flavivirus, Hepatitis C.

Example 8

The purpose of this experiment was to evaluate the efficacy ofsystemically administered squalamine as an antiviral treatment in asetting where the viral infection, such as Yellow Fever, has alreadybeen established.

Experimental design: Hamsters were randomly assigned to groups, with 10included in each and 20 placebo-treated controls. A 10⁻⁴ dilution(10^(2.0) CCID₅₀/ml) of the virus was prepared in minimal essentialmedia. Hamsters were injected into the peritoneum (i.p.) with 0.1 ml ofthe diluted virus (10 CCID₅₀/animal). Squalamine was administeredsubcutaneously (s.c.) at a total daily dose 15 mg/kg/d, given once a day(qd) beginning 24 h after introduction of virus and ending on 8 dayspost infection (dpi), 30 mg/kg/d, given once a day (qd) beginning 24 hafter introduction of virus and ending on 8 days post infection (dpi),and 30 mg/kg/d, given once a day (qd) beginning 48 h after introductionof virus and ending on 9 days post infection (dpi). Mortality wasobserved daily for 21 days, and weight was recorded on 0, 3, and 6 dpi.

Statistical analysis: Survival data were analyzed using the Wilcoxonlog-rank survival analysis and all other statistical analyses were doneusing one-way ANOVA using a Bonferroni group comparison (Prism 5,GraphPad Software, Inc).

Results: (FIG. 6) Whereas 100% of animals untreated died of Yellow Feverby day 11 post-infection, 60% of animals treated at 1 dpi with 15mg/kg/day (s.c.) squalamine for 8 days survived and appeared to be curedas measured by continued survival through day 21. Similarly, 60% ofanimals treated at 1 dpi with 30 mg/kg/day appeared to have been curedas indicated by survival through day 21. Treatment remained effectiveeven when dosing began on 2 dpi, with 40% of animals cured, when treatedwith squalamine at 30 mg/kg/day (s.c.) for 9 days.

The results of this example demonstrate that squalamine can be utilizedas an effective systemic antiviral therapy in already established viralinfection. Because of the similarity in the properties shared by theflavivirus family, in addition to Yellow Fever, squalamine could be usedto treat infections caused other members of the Flaviviridae including:Dengue, Hepatitis C, West Nile, Japanese Encephalitis, Tick borneEncephalitis, St. Louis Encephalitis, Murray Valley Encephalitis,Kyasanur Fever, and any novel as yet undiscovered virus classified as amember of the Flaviviridae.

Yellow fever virus utilizes a pH dependent entry pathway to initiateinfection. Based on the mechanism of action of squalamine and theefficacy of squalamine in the treatment of an established infectioncaused by Yellow fever, squalamine could be considered for the treatmentof other infections caused by viruses that utilize a pH dependent entrypathway such as members of the Orthomyxoviridae including: Influenza A,B, C, Isavirus, Thogotovirus; members of the Rhabdomyoviridae,including: Vesiculovirus, Lyssavirus, Cytorhabdovirus,Nucleorhabdovirus, Novirhabdovirus; members of the Adenoviridaeincluding: all Human Adenovirus types (1-55) and species (A-G),Atadenovirus, Avidenovirus, Icthadenovirus, Mastadenovirus,Siadenovirus; members of the Parvoviridae; members of the Filoviridae;members of the Iridoviridae; the Rubella virus.

Example 9

The purpose of this experiment was to evaluate the efficacy in an animalof systemically administered squalamine against a DNA virus. In thisexample mice have been infected with murine cytomegalovirus (MCMV), avirus similar to CMV that infects humans.

Experimental design: In this experiment mice were treated 24 hours priorto infection and treatment continued daily for 6 days post infection.Animals were sacrificed on days 3, 7, and 14, organs were harvested, andvirus content determined by a standard viral plaque assay. 54 maleBalb/c mice were inoculated into the peritoneum (i.p.) with 0.1 ml of avirulent strain of MCMV following a published protocol (Cavanaugh, Denget al. 2003). 18 animals received 5% dextrose i.p., and served ascontrols; 18 received 10 mg/kg/day of squalamine i.p., as a 1 mg/mlsolution in 5% dextrose; 18 received 10 mg/kg/day of squalamine s.c. Ondays 3, 7, and 14, 6 animals from each dosing cohort were randomlyselected, euthanized and viral titers determined from the liver, spleen,lung, and submaxillary gland.

Statistical analysis: Statistical analyses were done using one-way ANOVAusing a Bonferroni group comparison or via Student's T test (Prism 5,GraphPad Software, Inc).

Results: (FIG. 7) At day 3 a greater than 10-fold reduction in viralgrowth was observed in liver, spleen, and lung in animals that hadreceived squalamine via the i.p. route, with little effect observed inthe salivary gland. Between day 3 and 7, viral titers in spleen andliver fell about 10-fold in all groups, with the viral titers in thes.c. and i.p. groups significantly lower than in the control cohort; incontrast, viral titers increased between days 3 and 7 in the lungs andsalivary glands, with no significant differences observed betweengroups. By day 14, 8 days after squalamine had stopped, virus wasundetectable in the liver and spleen of animals that had receivedsqualamine i.p., and was significantly reduced in the s.c. treatmentgroup compared with controls. A trend toward reduction in viral titersin lung compared with control was observed in the i.p. treatment group.

The results of this example demonstrate that squalamine systemicallyadministered to an animal can effectively treat CMV infection and reduceviral titers to undetectable levels. Hence, squalamine can exhibitantiviral activity systemically against both RNA and DNA viruses.

This experiment also demonstrates that squalamine is active against amember of the Herpes Virus family, and supports its use in infectionscaused by other members of the Herpes family, including Humancytomegalovirus, Herpes Simplex 1, Herpes Simplex 2, Epstein Barr Virus,Varicella Zoster Virus, Roseolovirus (HHV6 and HHV7), Kaposi's SarcomaAssociated Herpes Virus, Cercopithecine herpesvirus-1, Murinegammaherpesvirus-68, the Bovine Herpesviridae, the Canine Herpesviridae,the Equine Herpesviridae, the Feline Herpesviridae, the DuckHerpesviridae, the Chicken Herpesviridae, the Turkey Herpesviridae,Porcine Herpesviridae and any as yet undiscovered virus subsequentlyclassified as a member of the Herpesviridae.

This experiment also demonstrates, by virtue of the measured reductionin viral titers within the spleen, that squalamine administeredsystemically can effectively render virally resistant the cells of thespleen that support CMV infection, which include macrophages. Thisresult supports the use of squalamine in the treatment of all viraldiseases in which the macrophage is subject to infection.

Example 10

The purpose of this experiment was to evaluate the efficacy ofsystemically administered squalamine against the Eastern EquineEncephalitis Virus, an RNA virus of the alphavirus family.

Experimental design: In this experiment hamsters (Syrian golden) weretreated with either 5% dextrose (n=10, each species) or 10 mg/kg/days.c. squalamine (n=10), beginning 24 hours prior to infection with EEEV,administered s.c., following a published protocol (Paessler, Aguilar etal. 2004). Treatments continued for 6 days after infection. Plasma viraltiters were measured, along with body weights and survival.

Statistical analysis: Survival data were analyzed using the Wilcoxonlog-rank survival analysis and all other statistical analyses were doneusing one-way ANOVA using a Bonferroni group comparison, or viaStudent's T test (Prism 5, GraphPad Software, Inc).

Results: Squalamine administration extended survival in the hamstercohort (FIG. 8). At the end of treatment, 6/10 hamsters receivingsqualamine were still alive, compared with 0/10 receiving vehicle.Concentration in the bloodstream of the hamster were determined (themice were not studied since squalamine was ineffective). Plasmaconcentrations of virus were lower by about 100 fold in hamsters thathad received squalamine compared with vehicle over the first 3 days postinfection, confirming that squalamine has antiviral activity in ananimal, likely the cause of improved survival (FIG. 9). We assume that amore pronounced effect on viremia would be observed with administrationof higher squalamine doses.

The results of this example demonstrate that squalamine can effectivelyreduce the concentration of virus in an animal when administeredsystemically. This experiment also demonstrates the activity ofsqualamine in treating an infection caused by a member of the Alphavirusfamily and supports its use in the treatment infection caused by othermembers of this family, including: Aura virus, Barmah Forest virus,Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equineencephalitis virus, Everglades virus, Fort Morgan virus, Getah virus,Highlands J virus Mayaro virus, Middelburg virus, Mosso das Pedras virus(78V3531, Mucambo virus, Ndumu virus, O'nyong-nyong virus, Pixuna virus,Rio Negro virus, Ross River virus, Salmon pancreas disease virus,Semliki Forest virus, Sindbis virus, Southern elephant seal virus,Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitisvirus, Western equine encephalitis virus, Whataroa virus, as well as anyas yet undiscovered virus subsequently classified as a member of theAlpaviridae.

Example 11

The purpose of this experiment was to demonstrate the antiviral effectsof squalamine against Dengue. Dengue is a flavivirus, related to YellowFever Virus, and human Hepatitis C virus. The study was conducted incell culture and utilized as substrate for infection a well studied lineof human endothelial cells (HMEC-1) anticipated to be responsive tosqualamine based on squalamine's known activity against endothelialcells.

Experimental design: Cells were grown on uncoated glass cover-slipsfollowing a published protocol (Zamudio-Meza, Castillo-Alvarez et al.2009). The cells were pretreated with squalamine for 2 hr at 37° C.prior to viral exposure; virus (multiplicity of infection of 3) remainedin contact with cells for 30 minutes at 4° C., followed by 90 minutes at37° C. The medium was then replaced with fresh medium lacking virus andsqualamine and maintained at 37° C. for 48 hrs. Cells were fixed andprocessed for immunohistochemical detection of viral E protein. Viral Eprotein expression was used to monitor the early stages of infection.

Results: Viral infection was markedly diminished at concentrations ofabout 40 μg/ml (80%), with almost 100% inhibition at 60 μg/ml and higher(FIG. 10). Cell viability was not diminished at the effective squalamineconcentrations, based on morphology and appearance under phase contrast.

This experiment demonstrates that squalamine has direct antiviralactivity against Dengue, a non-enveloped RNA virus of the flavivirusfamily. Because of the similarity in the properties shared by theflavivirus family, in addition to Yellow Fever and Dengue, squalaminewould be expected to be active against Hepatitis C, West Nile, JapaneseEncephalitis, Tick borne Encephalitis, St. Louis Encephalitis, MurrayValley Encephalitis, Kyasanur Fever, and any novel as yet undiscoveredvirus classified as a member of the Flaviviridae.

Example 12

The purpose of this experiment was to determine the antiviral activityof squalamine against human Hepatitis B virus. The experiment wasconducted in vitro, using primary human hepatocytes.

Experimental design: Primary human hepatocytes were established in a 96well microtiter plate. Squalamine was then added at concentrations of 2,6 or 20 μg/ml followed by an inoculum of Hep B virus, and then inculture for 16 hours, after which fresh medium was introduced, and thecells maintained in culture for 14 days. In a second experiment,squalamine at 6 or 20 μg/ml was added to the cells at 24 hours postinoculation for a 16 hour exposure, followed by removal of medium,replacement with fresh medium, and continued culture for 14 days. Viralgrowth was measured by PCR using viral specific primers and normalizedto the total RNA extracted from each corresponding well.

Results: Squalamine effectively inhibited HepB viral replication inhuman primary hepatocytes when added either during the initial exposureof virus to the cells, or at 24 hours. At a concentration of 20 μg/mlsqualamine inhibited viral production by 83% when added during initialstages of infection, and by 64% when added at 24 hours; at aconcentration of 6 μg/ml squalamine inhibited viral production by 54%when added at the onset of infection, and by 30% when added at 24 hours;squalamine at 2 μg/ml, added only at the outset of infection, inhibitedproduction by 14%.

The experiment demonstrates that squalamine can exert antiviral activityagainst a human Hepatitis B virus infection of human liver. Theexperiment demonstrates that squalamine can inhibit the early phase ofinfection as well as the production of virus of cells already infected.These data support the use of squalamine for the treatment of acute andchronic viral hepatitis caused by Hepatitis B.

Example 13

The purpose of this experiment was to determine the antiviral activityof squalamine against human Hepatitis Delta virus. HDV is a smallcircular RNA virus that causes hepatitis by itself or in conjunctionwith Hepatitis B virus. The experiment was conducted in vitro, usingprimary human hepatocytes.

Experimental design: Primary human hepatocytes were established in a 96well microtiter plate. Squalamine was then added at concentrations of 20or 60 μg/ml followed by an inoculum of Hep D virus, and then in culturefor 3 hours, after which fresh medium was introduced, and the cellsmaintained in culture for 7 days. Viral growth was measured by PCR usingviral specific primers and normalized to the total RNA extracted fromeach corresponding well.

Results: Squalamine effectively inhibited Hepatitis Delta viralreplication in human primary hepatocytes when added during the initialexposure of virus to the cells. At a concentration of 20 μg/mlsqualamine inhibited viral production by 90% when added during initialstages of infection. The 60 μg/ml concentration proved to be cytotoxicin vitro.

The experiment demonstrates that squalamine can exert antiviral activityagainst a human Hepatitis Delta virus infection of human liver. Thesedata support the use of squalamine for the treatment of acute andchronic viral hepatitis caused by Hepatitis Delta virus. Since HepatitisB and D frequently co-infect the same individual, these data wouldsupport use of squalamine for the treatment of both infectionsconcurrently.

Squalamine inhibits the replication of both Hepatitis B virus andHepatitis D virus in primary human hepatocytes, two viruses that differin their structure, mode of entry, and replicative biology, a resultanticipated by the proposed antiviral mechanism of squalamine. Theseresults strongly suggest that squalamine should be effective againstother viral infections of the human liver caused by the common Hepatitisviruses: Hepatitis A virus, Hepatitis E, Hepatitis F and Hepatitis G,and any other viral infection of the hepatocyte.

Example 14

The purpose of this experiment was to determine the antiviral activityof squalamine against human immunodeficiency virus (HIV).

Experimental design: A line of HeLa cells is utilized that expresses thereceptors and co-receptors required for the binding and entry of HIV,namely CXCR4, CD4 and CCR5. The HeLa line also has a luciferase reportergene driven by the HIV-LTR so if infection occurs luciferase isexpressed (Harmon, Campbell et al.). The amount of luciferase made ismeasured and serves as a measure of infection. The cells are plated andinfected in the presence or absence of the squalamine. A strain ofvesicular stomatitis virus (VSV) that contains HIV genes required toactivate the luciferase gene is utilized as a toxicity control, andshould infect the cells equally well with or without the squalamine(unless squalamine also inhibits the infectivity of VSV). Once the cellsare infected they incubate for at least 1 day (in the presence of thedrug). The virus and drug are removed from the cells and then the cellsare lysed with PBS+0.2% TritonX-100. Luciferase assays are done on thelysates to measure the level of infection.

Results: Squalamine inhibited HIV infection by about 50% at aconcentration of 30 μg/ml compared with vehicle alone, with no evidenceof toxicity apparent. At 20 μg/ml inhibition of about 20% was observed.

These data support the use of squalamine for the treatment of HIV andother retroviral infections. In addition these data demonstrate thatsqualamine can block the infectivity of enveloped viruses that entercells via a pH independent fusion process. Thus, these data support theuse of squalamine in the treatment of viral infections caused by virusessuch as the retroviridae and the paramyxoviridae, including: Newcastledisease virus, Hendravirus, Nipah virus, measles virus, Rinderpestvirus, Canine distemper virus, Sendai virus, Human parainfluenza 1, 2,3, 4, mumps virus, Menangle virus, Tioman virus, Tuhokovirus 1, 2, 3,Human respiratory syncytial virus, avian pneumovirus, humanmetapneumovirus; viruses such as the picornaviridae, including: Humanenterovirus A, B, C, D, Human rhinovirus A, B, C, Encephalomyocarditisvirus, Theilovirus, Foot and mouth virus, Equine rhinitis A virus,Bovine Rhinitis B virus, Hepatitis A virus, Human Parechovirus, Ljunganvirus, Aichi virus, Teschovirus, Sapeloviris, Senecavirus, Tremovirus,Aviheptovirus; viruses such as the rotoviridae, including: rotavirus A,B, C, D, E; viruses such as the papovaviridae.

* * *

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention. Thus, it is intended that the present invention cover themodifications and variations of this invention, provided they comewithin the scope of the appended claims and their equivalents.

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What is claimed is:
 1. An in vivo method of treating a viral infectionin a mammal comprising administering a composition comprising: (a) apharmaceutically acceptable grade of squalamine or a pharmaceuticallyacceptable salt thereof in an amount sufficient to produce an antiviraleffect, and (b) at least one pharmaceutically acceptable carrier,wherein the composition is systemically administered via a methodselected from the group consisting of intravenously, subcutaneously,intramuscularly, and orally; and wherein the viral infection is causedby Hepatitis B virus.
 2. An in vivo method of treating a viral infectionin a mammal comprising administering a composition comprising: (a) apharmaceutically acceptable grade of squalamine or a pharmaceuticallyacceptable salt thereof in an amount sufficient to produce an antiviraleffect, and (b) at least one pharmaceutically acceptable carrier,wherein the composition is systemically administered via a methodselected from the group consisting of intravenously, subcutaneously,intramuscularly, and orally; and wherein the viral infection is causedby Hepatitis Delta virus.
 3. An in vivo method of treating a viralinfection in a mammal comprising administering a composition comprising:(a) a pharmaceutically acceptable grade of squalamine or apharmaceutically acceptable salt thereof in an amount sufficient toproduce an antiviral effect, and (b) at least one pharmaceuticallyacceptable carrier, wherein the composition is systemically administeredvia a method selected from the group consisting of intravenously,subcutaneously, intramuscularly, and orally; and wherein the viralinfection is caused by Hepatitis C virus.
 4. An in vivo method oftreating a viral infection in a mammal comprising administering acomposition comprising: (a) a pharmaceutically acceptable grade ofsqualamine or a pharmaceutically acceptable salt thereof in an amountsufficient to produce an antiviral effect, and (b) at least onepharmaceutically acceptable carrier, wherein the composition issystemically administered via a method selected from the groupconsisting of intravenously, subcutaneously, intramuscularly, andorally; and wherein the viral infection is caused by Dengue virus.